A.

Prokop - A rough guide to Drosophila mating schemes 1

A rough guide to Drosophila mating schemes (version 1.2) 1

1. Why work with the fruitfly Drosophila melanogaster?

More than a century ago the fruitfly Drosophila melanogaster was introduced as the invertebrate
model organism that founded the field of classical genetics. It has been argued that Drosophila, as
an omnipresent follower of human culture, was easy to obtain and maintain in laboratories, and that
it was kept in many laboratories as a cheap model for student projects suitable in times of neo-
Darwinism (the study of Darwinian evolution with Mendelian genetics) [1]. Several laboratories
started using the fly for their main research, but it was the serendipitous discovery of the white
mutation and recognition of its linkage to the X chromosome in 1910 by T.H. Morgan which kick-
started the systematic use of the fly for genetic research, essentially fuelled by Morgan's graduate
2
students Sturtevant and Bridges [1,7] . Building on the sophisticated fly genetics gained during the
early decades, research during the second half of the 20th century gradually turned flies into a
powerful "boundary object" linking genetics to other biological disciplines [10]. Thus, fly genetics
was systematically applied to the study of development, physiology and behaviour, generating new
understanding of the principal genetic and molecular mechanisms underpinning biology, many
being conserved with higher animals and humans [7,10,11,12,13,14,15]. Notably, it has been
estimated that “...about 75% of known human disease genes have a recognisable match in the
genome of fruit flies” [17]. Therefore, besides remaining a powerhouse for unravelling concepts and
fundamental understanding of basic biology, Drosophila is nowadays often used as a “test tube” to
screen for genetic components of disease-relevant processes or pathways, or to unravel their
cellular and molecular mechanisms, covering a wide range of disease mechanisms including
3
neurodegeneration and even neurotoxicology [18,19,20,21] . It is therefore not surprising that
Drosophila is the insect behind six Nobel laureates (Box 1).

Box 1. Nobel prizes for work on Drosophila (www.nobelprize.org/nobel_prizes/medicine/laureates/)

1933 Thomas Hunt Morgan - the role played by chromosomes in heredity
1946 Hermann Joseph Muller - the production of mutations by means of X-ray irradiation
1995 Edward B. Lewis, Christiane Nüsslein-Volhard, Eric F. Wieschaus - the genetic control of early
embryonic development
2011 Jules A. Hoffmann - the activation of innate immunity

Drosophila's enormous success originates from the numerous practical advantages this tiny insect
and the community of fly researchers have to offer to the experimenter. The most important
advantages are briefly listed below:

1
Updated versions of this document can be downloaded @ dx.doi.org/10.6084/m9.figshare.106631
2
Dan Lindley (2008) Drosophila genetics - The first 25 years @ hstalks.com/?t=BL0341788
3
Informative lay descriptions of fly research can be found on the Wellcome Trust Blog:
The portrait of a fly (Part 1) - wellcometrust.wordpress.com/2012/11/20/feature-the-portrait-of-a-fly-part-1/
The portrait of a fly (Part 2) - wellcometrust.wordpress.com/2012/11/23/the-portrait-of-a-fly-part-2-fly-on-the-wall/
A. Prokop - A rough guide to Drosophila mating schemes 2

 Fruit flies are easy and cheap to keep. High numbers of different fly stocks can be kept in a
handful of laboratory trays, thus facilitating high-throughput experiments and stock
management (section 3).
 A fruit fly generation takes about 10 days (Fig.1), thus fly research progresses rapidly.
Pedigrees over several generations can be easily planned and carried out in a few months.

Figure 1. The life cycle of Drosophila melanogaster

Fertilised females store sperm in their receptaculum
seminis for the fertilisation of hundreds of eggs to be laid
over several days. At 25°C embryonic development lasts
for ~21hr. The hatched larvae (1st instar) take 2 days to
molt into 2nd then 3rd instar larvae. 3rd instar larvae
continue feeding for one more day (foraging stage) before
they leave their food source and migrate away
(wandering stage) and eventually pupariate (prepupa
then pupa). During the pupal stages, all organs
degenerate (histolysis) and restructure into their adult
shapes (metamorphosis). 10d after egg-lay, adult flies
emerge from the pupal case. Newly eclosed males
require up to 8 hr to mature sexually, facilitating the
collection of virgin females (section 3). The times
mentioned here need to be doubled when flies are raised
at 18°C [3]. Image modified from FlyMove [22].

Figure 2 A typical flow diagram of how genetic

screens in Drosophila contribute to research
A) To induce random mutations, large numbers of
flies are manipulated chemically (e.g. using EMS,
ethyl methanesulfonate - highly carcinogenic!),
genetically (e.g. through P-element mutagenesis;
section 5.1) or with irradiation (e.g. applying X-
ray). Other unbiased approaches are screens with
large collections of transgenic RNAi lines to
systematically knock down genes one by one
(section 5.2e) or with EP-line collections to
systematically over-express genes (section 5.2c).
B) The essential task is to select those mutant or
genetically manipulated animals that display
phenotypes representing defects in the biological
processes to be investigated. C) The responsible
gene is either pinpointed by the specific RNAi- or
EP-line inducing the phenotype, or classical
genetic or molecular strategies are used to map
newly induced mutations to defined genes within
the fly genome (Fig. 12B and section 6). D) Once
the gene is identified, its nature and normal
function can be studied. E) Using the gene's
sequence in data base searches (capitalising on
the existing sequences of total genomes)
homologous genes in higher vertebrates or humans are identified. Based on knowledge derived from fly
research and the empirical assumption that principal mechanisms are often conserved, informed and
focussed experiments can be carried out on these genes in vertebrate/mammalian model organisms, or
human patients can be screened for mutations in these genes.

 The fly genome is of low redundancy, i.e. only one or very few genes code for members of the
same protein class. In contrast, higher organisms usually have several paralogous genes
coding for closely related proteins that tend to display functional redundancy and complicate
loss-of-function analyses.
A. Prokop - A rough guide to Drosophila mating schemes 3

 A particular strength of Drosophila is the possibility to perform unbiased screens for genes
that regulate or mediate biological processes of interest, often referred to as forward genetics
(Fig. 2; Box 2). Highly efficient and versatile strategies have been developed that can be
adapted to the experimenter's needs [23,24,25,26,27].
 Virtually every gene of Drosophila is amenable to targeted manipulations through a wide
range of available genetic strategies and tools, ideal to perform reverse genetics (Box 2)
1
[5,28,29,30,31,32,33,34] .
 Experimental manipulations and observations of cells and tissues are relatively easy. Thus,
organs are of low complexity and size, and can often be studied live or via straightforward
fixation and staining protocols in the whole organism. These experiments are usually not
subject to legal requirements or formal procedures.
 More than a century of fly work has produced a huge body of knowledge and a rich resource
of genetic tools. Well organised databases and stock centres provide easy access to both
knowledge and genetic tools [36,37]. Furthermore, the highly collaborative spirit of the fly
community that has prevailed since the early days of fly research [1], enormously facilitates
research through generous exchange of materials and information.

Box 2. Concepts for genetic research: LOF versus GOF, forward versus reverse genetics
Two principal classes of manipulation are usually employed to study gene function. LOSS-OF-
FUNCTION (LOF) approaches attempt to eliminate gene function partially or completely, for example by
employing LOF mutant alleles (section 4.1.2), knock-down of genes using RNA interference strategies
(section 5.2e), the targeted expression of dominant-negative constructs (e.g. catalytically dead versions
of enzymes titrating out the function of the endogenous healthy enzyme), or transgenic expression of
single-domain antibodies [4]. GAIN-OF-FUNCTION (GOF) approaches attempt to obtain functional
information by creating conditions where the gene is excessively or ectopically expressed or its function
exaggerated. This can be achieved through targeted over-expression of genes, either of their wild type
alleles or of constitutive active versions (section 5), or through the use of GOF mutant alleles (section
4.1.2).
Gene manipulations are generally employed to serve two principal strategies. FORWARD
GENETICS is the approach to identify the gene(s) that are responsible for a particular biological process
or function in an organism. In Drosophila this is usually performed through using unbiased large-scale
LOF or GOF screens to identify genes that can disturb the process/function in question (Fig. 2).
REVERSE GENETICS is the approach to unravel the functions behind specific genes of interest, for
example when trying to understand molecular mechanisms or functions of genes known to cause human
disease (using the fly as a "test tube"). For this, LOF or GOF approaches are employed, using mutant
alleles or genetic tools that are often readily available or can be generated. The generation of transgenic
tools is daily routine in most fly laboratories (section 5.1). Also manipulations of genes in situ, i.e. in their
chromosomal location, can be achieved through various strategies, such as
 classical mutagenesis strategies to generate candidate alleles that are then selected over suitable
deficiencies uncovering the targeted gene locus (section 6c)
 generation of targeted deletions at the gene locus through mobilising local P-elements (section 5.1)
 targeted manipulations of the gene locus through genomic engineering using recombinase-based
strategies [9] or TALEN strategies (transcription activator-like effector nuclease) [16]

2. The importance of genetic mating schemes

Daily life in a fly laboratory requires performing classical genetic crosses. In these crosses, mutant
or genetically modified flies are used (Box 3). These different fly variants are the bread-and-butter
of fly research, providing the tools by which genes are manipulated or visualised in action in order
to investigate their function. The art of Drosophila genetics is to use these tools, not only in isolation
but often combined in the same flies. This combinatorial genetic approach significantly enhances
the information that can be extracted.

1
for overviews of Drosophila genetics see http://www.scribd.com/doc/6125010/Drosophila-as-a-Model-Organism and [35]
(http://highered.mcgraw-hill.com/sites/007352526x/student_view0/genetic_portrait_chapters_a-e.html)
A. Prokop - A rough guide to Drosophila mating schemes 4

Box 3. Fly stocks available for Drosophila research comprise...

1. ...flies carrying classical loss- and gain-of-function mutations or deficiencies (section 4.1.b)
2. ...flies with chromosomal rearrangements (duplications, inversions, translocations etc.) [2,3]
3. ...flies with balancer chromosomes (section 4.3)
4. ...flies with transgenic constructs encoding a range of products (section 4.4.) including..
o ..reporter genes, such as lacZ or fluorescent proteins, fused to gene-specific or inducible
promoters, or under the control of position-specific activating elements at their chromosomal
insertion site
o ..wildtype or mutant versions of genes from Drosophila or other organisms
o ..exogenous transcription factors (e.g. Gal4, tTA) with known expression patterns to induce
targeted expression of a gene of choice
o ..small interfering RNAs to knock down gene expression
o ..recombinases (e.g. flippase, ϕC31) or their recombination target sites at specific chromosomal
locations (e.g. FRT or attP); they are jointly used for site-directed insertion of transgenes or to
generate mosaics of mutant cells in the germline or in somatic tissues
o ..genetically encoded toxins (e.g. ricin, tetanus toxin), optogenetic tools (e.g. channel rhodopsin,
Ca2+ indicators) or other physiological tools (e.g. Kir channels, Shibirets) for the analysis or
experimental manipulation of cells
o ..whole chromosomal fragments for rescue, gain-of-function or targeted mutagenesis experiments
[5,6]

For example, you investigate a certain gene called Mef2. You have isolated a candidate
mutation in this gene which, when present in two copies in embryos, correlates with aberrant
muscle development. You hypothesise that this phenotype is caused by loss of Mef2 function. A
standard approach to prove this hypothesis is to carry out "rescue experiments" by adding back a
wild type copy of the gene into the mutant background, analogous to gene therapy. For this, you will
need to clone the Mef2 gene and generate transgenic fly lines for the targeted expression of Mef2
(section 5.1). To perform the actual experiment, you now need to bring the Mef2 transgenic
construct into Mef2 mutant individuals. This last step requires classical genetic crosses and the
careful design of genetic mating schemes.
These mating schemes are a key prerequisite for successful Drosophila research. The rules
underpinning these schemes are simple. Yet, they often require thinking ahead for several
generations, comparable to planning your moves during a game of chess. To enable you to design
such mating schemes, this manual will provide you with the key rules of the game and explain the
main parameters that need to be considered.

3. How to handle flies in the laboratory

Before starting the theoretical part, it is necessary to give a brief insight into the practical aspects of
fly husbandry and how the genetic crosses are performed. This should allow you to imagine the
actual "fly pushing" work required to execute the mating schemes designed on the drawing board.
As indicated in Box 3, many different fly stocks are available for fly work. Drosophila
research groups usually store in their laboratories considerable numbers of stocks relevant to their
projects (Fig. 3A). In this way stocks are readily available to kick-start practical work on
experimental ideas that arise through daily discussion and thought. Other stocks can be ordered
from public or commercial stock centres (FlyBase / Resources / Stock Collections) or by sending
requests to colleagues all over the world most of whom are willing to freely share fly stocks once
published in scientific journals. Note, that new flies coming into the laboratory should be kept in
quarantine and observed for a couple of generations in order to exclude diseases or parasites they
may carry. Fly stocks are kept in small vials containing larval food and they can easily be
transferred to fresh vials for maintenance (Fig. 3B). These vials are usually stored in trays in
temperature-controlled rooms or incubators (Fig. 3A). As indicated in Fig. 1, temperature influences
1
the developmental time of flies .

1
detailed stock-keeping instructions: flystocks.bio.indiana.edu/Fly_Work/culturing.htm
A. Prokop - A rough guide to Drosophila mating schemes 5

Figure 3. Maintaining and handling flies in the laboratory

1
A) Large numbers of different fly stocks are stored in trays in temperature controlled rooms or incubators
(the trays shown here each hold two copies of 50 stocks). B) Each fly stock is kept in glass or plastic vials
containing food, the main ingredients of which are corn flour, glucose, yeast and agar. The vials are closed
with foam, cellulose acetate, paper plugs or with cotton wool. Larvae live in the food. When reaching the
wandering stage they climb up the walls (white arrow) where they subsequently pupariate (white arrow
head). C-E) To score for genetic markers and select virgins and males of the desired phenotypes, flies are
immobilised on CO2-dispensing porous pads (E), visualised under a dissecting scope (C, D) and
eventually disposed of into a morgue or transferred to fresh vials using a paint brush, forceps or aspirator
(pooter) (C, E).

Tip 1. Keeping information about laboratory stocks

Work in a fly laboratory involves constant influx of new fly stocks, but only a small percentage of these will
eventually be kept in your stock collection. Follow good practice by making it your routine to instantly
document the essential information for each incoming stock in a dedicated folder or data sheet/base
before it gets lost and forgotten in daily routine:
1. Keep the full original genetic description and any other information you may find on vials or
accompanying notes (e.g. stock centre references or any other seemingly meaningless numbers).
Note that genetic descriptions you are given by the donor may be incomplete, and your accessory
notes may provide unique identifiers for this fly stock when communicating with the donor
laboratory.
2. Note down the donor laboratory/person and contact. You will need this information for further
enquiries and acknowledgements in future publications.
3. When introducing stocks into your collection, transfer the above information into the accompanying
data base/sheet. Make sure there is a proper genetic description, a clearly assigned short hand for
daily use, info on the donor and the key reference publication. This information will be most useful
when writing up your project and for people succeeding you in the laboratory.

 Stock keeping is usually done at 18°C (generation time of about 1 month). Be aware that you
deal with live animals that need to be cared for like pets! It is good practice to keep one young
and one two week older vial of each stock. Every fortnight, freshly hatched flies from the month
old vial are flipped into a fresh vial, whilst the two-week old vial should have produced larvae
and serves as a back-up. Such a routine allows you to spot any problems on time, such as
infections (mites, mould, bacteria, viral infections) [3], the need to add water (if the food is too
dry) or to reduce humidity (if vials are too moist).

1
Incubators need to be fly-proof: copper is aggressively corroded in the presence of flies and either needs to be well
protected (e.g. coated with resin) or should be avoided and replaced by stainless steel.
A. Prokop - A rough guide to Drosophila mating schemes 6

 Experiments with flies tend to take place at room temperature or at certain conventional
temperatures, such as 25°C for well timed experiments (Fig. 1) or 29°C to speed up
development or enhance targeted gene expression with the Gal4/UAS system (section 5.2).

Figure 4. Criteria for gender selection

Images show females (top) and males (bottom): lateral whole body view (left), a magnified view of the front
legs (2nd column), dorsal view (3rd column) and ventral view (right) of the abdomen. Only males display sex
combs on the first pair of legs (black arrow heads). Females are slightly larger and display dark separated
stripes at the posterior tip of their abdomen, which are merged in males (curved arrows). Anal plates (white
arrows) are darker and more complex in males and display a pin-like extension in females. The abdomen
and anal plate are still pale in freshly eclosed males and can be mistaken as female indicators at first sight.
Photos are modified from [38] and [39]. During a very short period after eclosion, females display a visible
dark greenish spot in their abdomen (meconium; not shown) which is a secure indicator of virginity even if
fertile males are present.

To perform crosses, females and males that carry the appropriate genotypes are carefully
selected. Some aspects need consideration:
 Males and females need to be distinguished using the criteria explained in Figure 4.
 Selected females have to be virgin, i.e. selected before they are randomly fertilised by sibling
males in their vial of origin. To select virgins, choose vials containing many dark mature pupae
from which adult flies are expected to eclose. To start the selection procedure, discard all flies
from the vial and thoroughly check that all eclosed flies (including those that transiently stick to
the food or walls) have been removed or otherwise eliminated. The key rationale of this
procedure is that freshly eclosed males remain sterile for a period of several hours and will not
court females. Hence, after clearing vials, all females eclosed within this period will be virgin.
This period lasts for 5-8 hrs at 25°C, about double the time at 18°C, and considerably longer at
even lower temperatures (we use 11°C to maintain crosses up to two days for subsequent virgin
collection). Therefore, a typical routine for virgin collection is to keep vials at low temperatures
overnight (ideally below 18°C) and harvest virgins first thing in the morning. During the day,
they are kept at higher temperatures (to enhance the yield) and harvested again around
lunchtime and early evening, before moving them back to lower temperature for the night.
 Flies have to be selected for the right phenotypic markers. When designing a mating
scheme, combinations of markers need to be wisely chosen so that the correct genotypes of
both sexes can be unequivocally recognised at each step of the mating scheme (often from
parallel crosses). Genetic markers will be explained in section 4.2., and the rules how to choose
them will become clear from later sections.
To select them for gender and phenotypic markers, freshly eclosed flies are tipped from their vial
onto a porous pad dispensing CO2. CO2 acts as a narcotic and is not harmful if exposure is kept to a
few minutes. Flies can be easily inspected on this pad under a dissection microscope (Fig. 3C-E).
Selected flies are added to fresh standard vials properly labelled with gender and genotype (Fig.
3B) and kept at standard temperature (room temperature or 25°C). Remaining flies are disposed of
in a fly morgue (usually a bottle containing 70% alcohol) and never returned to their vials of origin.
Some further considerations are explained in the box "Tip 2"
A. Prokop - A rough guide to Drosophila mating schemes 7

In general, more female flies are used in a cross than male flies, with two thirds being
female as a reasonable approximation (unless males are expected to be of low fitness due to the
mutations they carry). Also, if gender choice is an option and one of the stocks/genotypes to be
used is morbid, choose the more vital stock/genotype for virgin collection. In general, consider that
di- and trihybrid crosses (see example in Fig. 6) and crosses with mutant combinations that affect
viability will have a very low yield of the required offspring and have to be initiated by large volume
crosses. Consequently, expect that the volume of flies available for crosses in a complex mating
scheme may gradually reduce from generation to generation. Also be aware that certain genotypes
may cause flies to eclose later or earlier than others. For example, males carrying the balancer
chromosome FM7 in hemizygosis (over a Y chromosome) may eclose days after their female
siblings carrying the same balancer in heterozygosis (over an X chromosome; see Fig. 10). Finally,
fly strains may be carrying bacterial or viral diseases or they can be infected with fungi or mites [3].
These conditions can pose a threat to the feasibility of mating schemes. The best prophylaxis is
careful and regular husbandry of your fly stocks.
Especially in complex mating schemes with complex marker combinations, a safe way of
selecting the right animals for your next cross is to merely separate males from females into distinct
vials during your daily routine. Only when enough animals have been collected, perform the marker
selection in one single session. This mode is safer and less time-consuming, especially for the
inexperienced fly pusher or when various crosses are running in parallel and keeping an overview
becomes a challenge.

Tip 2. How to perform counts on experimental crosses

Certain experiments demand that you quantitatively assess the relative abundance of the various geno-/
phenotypes emerging from a cross, for example when carrying out meiotic mapping experiments
(section 6b). In another scenario, a mutant allele may cause death in some of the individuals of a relevant
genotype but not in all of them (semi- or sub-lethal allele). To determine the degree of lethality (as one
possible measure for the strength of your mutant allele), you need to perform geno-/phenotypic counts of
homozygous mutant versus heterozygous/balanced animals.
1. To achieve accuracy of results and not bias the outcome, you need to make sure that vials are not
over-populated with larvae of the F1 generation (i.e. the individuals that will be assessed as
adults). Over-population tends to disadvantage morbid individuals, enhancing their lethality above
usual proportion.
a. Make sure you transfer parents to new vials when sufficient eggs have been laid (within a time
frame of 1 day to 1 week, depending on fertility of stocks used and number of parental flies).
b. Monitor crosses for start of eclosure, then score crosses daily, otherwise sick eclosed progeny
may get stuck and lost in the wet food.
c. Weakest animals tend to hatch late, therefore continue scoring for as long as possible.
However, be aware that F2 flies start emerging after about 19 days in a modestly populated
tube at 25ºC (Fig. 1). When this point is reached cease counting and discard the tube.
2. When scoring a large number of flies, arrange them into a line across the plate and pull out one
phenotypic class at a time. In this way you only have 2 piles of flies on the plate at any one time.

4. How to design a mating scheme

4.1. Genetic rules
In order to design mating schemes for Drosophila, the typical rules of classical genetics can be
applied. These rules are briefly summarised here and are described in greater depth elsewhere
[2,3].

4.1.1. Law of segregation

Drosophila is diploid, i.e. has two homologous sets of chromosomes, and all genes exist in two
copies (except X-chromosomal genes in males; Fig. 5). By convention, homologous alleles are
separated by a slash or horizontal line(s) (Fig. 6, Box 5). According to the first law of Mendel (law of
segregation), one gene copy is inherited from each parent. The two copies of a gene are
separated during meiosis and only one copy is passed on to each offspring (Fig. 6). Rare
exceptions to this in which both copies pass to one gamete are termed non-disjunction events.
A. Prokop - A rough guide to Drosophila mating schemes 8

Figure 5. Drosophila chromosomes

Cytological images of mitotic Drosophila chromosomes. Left: Female and male cells contain pairs of
heterosomes (X, Y) and three autosomal chromosomes. Right: Schematic illustration of Drosophila
salivary gland polytene chromosomes which display a reproducible banding pattern used for the
cytogenetic mapping of gene loci (black numbers; see FlyBase / Tools / Genomic/Map Tools /
Chromosome Maps for detailed microscopic images); 2nd and 3rd chromosomes are subdivided into a left
(L) and right (R) arm, divided by the centrosome (red dot). Detailed descriptions of Drosophila
chromosomes can be found elsewhere [40].

1
4.1.2. Alleles
Genes exist in different alleles. Most loss-of-function mutant alleles (hypo- or amorphic/null) are
recessive. Their phenotypes are not expressed in heterozygous (-/+) but only in homozygous
animals (-/-), i.e. the wildtype allele mostly compensates for the functional loss of one gene copy
(see w, vg or e in Fig. 6). Loss-of-function mutant alleles can also be dominant. For example,
phenotypes are observed in animals heterozygous for Ultrabithorax (Ubx/+), Polycomb (Pc/+), or
Notch (N/+) loss-of-function alleles, i.e. the wildtype allele is insufficient to compensate for loss of
one functional gene copy (haplo-insufficiency). Dominant alleles can also be gain-of-function,
usually caused by over-expression of a gene product (hypermorph or "dominant negative"
antimorph) or by ectopic expression or activation of a gene product, potentially conveying novel
gene functions (neomorph). For example, BarH1 over-expression in the eye causes kidney-shaped
eyes in Bar1/+ individuals (Fig. 6) [41], ectopic Antp expression in antennae the antenna-to-leg
transformations in Antp73b/+ (Fig. 9) [42], and Krüppel mis-expression the reduced eyes in If1/+
animals (Fig. 9) [43]. Dominant alleles may display intermediate inheritance showing a stepwise
increase in phenotype strength from heterozygous to homozygous animals. Thus, the eyes of
heterozygous flies (B1/+) are kidney-shaped, whereas they display a stronger slit-shaped phenotype
in homo- (B/B) or hemizygous (B/Y) flies (Fig. 6). Animals carrying the loss-of-function mutant allele
abd-AMX1 in heterozygosis are viable and show a weak dominant cell proliferation phenotype,
whereas homozygous animals are lethal and show a strong cell proliferation phenotype [8]. Note,
that the phenotype distribution in pedigrees involving dominant mutant alleles differs from those
with recessive mutant alleles (Fig. 6). Also note that the existence of dominant and recessive alleles
has impacted on gene names (capitalisation of the first letter), which can be confusing or even
misleading (Box 4).

4.1.3. Independent assortment of chromosomes

Drosophila has one pair of sex chromosomes (heterosomes: X/X or X/Y) and three pairs of
autosomes (Fig. 5). Usually, non-homologous chromosomes behave as individual entities during
meiosis and are written separated by semicolon in crossing schemes (Fig. 6, Box 5). According to
the second law of Mendel (law of independent assortment), they assort independently of one
another during gamete formation, leading to a high number of possible genotypes (Fig. 6). A good
strategy to deal with this complexity during mating scheme design is to define selection criteria for
each chromosome independently (curly brackets in Fig. 6; see Box 5). The 4th chromosome
harbours very few genes and its genetics slightly differs from other chromosomes [2]. It plays a
negligible role in routine fly work and will therefore not be considered here.
1
see also http://en.wikipedia.org/wiki/Muller's_morphs
A. Prokop - A rough guide to Drosophila mating schemes 9

Figure 6. Independent assortment of alleles & comparison of recessive and dominant inheritance
Two examples of crosses between heterozygous parents (P) involving recessive alleles (top left) and a
dominant allele (green box top right) are shown. Homologous alleles are separated by a horizontal line;
maternal alleles are shown in black, paternal ones in blue. Mutant alleles are w (white; white eyes), vg
(vestigial; reduced wings), B (Bar; reduced eyes); phenotypes are indicated by fly diagrams (compare Fig.
9). When comparing inheritance of the eye marker mutations w (left) and B (right), it becomes apparent
that the allele assortments are identical, yet only the heterozygous B mutant females show an intermediate
eye phenotype.
The left example is a dihybrid cross involving mutant alleles on X and 2nd chromosomes (separated
by semicolons). In the first offspring/filial generation (F1) each chromosome has undergone independent
assortment of alleles (demarcated by curly brackets) and each of the four possible outcomes per
chromosome can be combined with any of the outcomes of the other two chromosomes resulting in 4 x 4 =
16 combinations. In case of two autosomal genes, the phenotypic distribution would be 9:3:3:1
(homogeneously coloured fields in the Punnett square), as compared to 3:1 in a monohybrid cross (only
one of 4 animals displays vg phenotype). However, since w is X-chromosomal, the phenotypic distribution
here is 6:6:2:2 (indicated by hatched fields in Punnett square). The Punnett square lists all possible
combinations (symbols explained on the right); red and blue stippled boxes show the same examples of
two possible offspring in both the curly bracket scheme and the Punnett square. Note that the Punnett
square reflects the numerical outcome of this cross in its full complexity, whereas the curly bracket strategy
only qualitatively reflects potential combinations and is easier to interpret for the purpose of mating scheme
design (Box 5). The complexity of Punnett squares become even more obvious when dealing with trihybrid
crosses (Appendix 2).

4.1.4. Linkage groups and recombination

Genes located on the same chromosome are considered a linkage group that tends to segregate
jointly during meiosis. However, when homologous chromosomes are physically paired during
meiotic prophase (synapsis), the process of intra-chromosomal recombination (crossing-over)
can lead to exchange of genetic material between homologous chromosomes (Fig. 7; note, that
recombination does not occur on the 4th chromosome). As a rule of thumb, the recombination
frequency increases with distance between gene loci, but non-uniformly across the chromosome
arms (map expansion/compression). Therefore, frequencies are high in the middle of
chromosome arms and low in regions adjacent to heterochromatin-rich telomeres and centromeres.
A. Prokop - A rough guide to Drosophila mating schemes 10

Recombination frequencies have been used to generate spatial chromosomal maps of gene loci
(recombination maps), defining 1% chance of crossing-over between two loci as 1 map unit (or
centimorgan, cM) [2]. 50% is the maximum detectable crossing-over frequency because crossing-
over is happening at the 4-strand stage; only 2 strands are involved in any one event and exchange
between sister chromatids produces no observable changes. If two genes are 50 cMs apart then
they are equivalent to being unlinked (due to the increase in multiple crossing-over events occurring
between them). If the location of two loci is known relative to the cytogenetic map, their position on
the recombination map can be roughly estimated and the recombination frequency between them
deduced (Fig. 7B and bottom of Box 4).

Figure 7. Inheritance of genes on the same chromosome (linked genes)

(P) A cross between flies heterozygous for viable recessive mutations of the 3rd chromosomal loci rosy
(ry; brownish eyes, 87D9-87D9) and ebony (e; black body colour, 93C7-93D1); female chromosomes are
shown in green, male in blue. According to the law of segregation, homologous chromosomes are
distributed to different gametes (egg and sperm) during gametogenesis. Male chromosomes do not
undergo crossing-over. In females, crossing-overs are possible (red zigzag lines), and recombination
between any pair of genes may (yes) or may not (no) occur (at a frequency dependent on their location
and distance apart), thus increasing the number of different genotypes. In the first filial generation (F1),
three potential genotypes and two potential phenotypes would have been expected in the absence of
recombination (strict gene linkage); this number is increased to 7 genotypes and 4 phenotypes when
including crossing-over.

For mating schemes, recombination can be a threat as well as an intended outcome:

 There are two key remedies to prevent unwanted recombination during mating schemes. The
first strategy is to use balancer chromosomes (section 4.3). The second strategy is to take
advantage of the recombination rule. The recombination rule states that there is no
crossing-over in Drosophila males (Fig. 7). The reason for this is not clear but might relate
to the observation that, although reductional divisions occur and haploid gametes are
produced, the type of genes expressed in male meiosis "is much more similar to mitosis than to
female meiosis" [44].
 In other occasions it can be the intended outcome of a mating scheme to recombine
mutations onto the same chromosome. For example, in reverse to what is shown in Fig. 7,
you may want to combine the rosy (ry) and ebony (e) mutations from different fly stocks onto
one chromosome in order to perform studies of ry,e double-mutant flies. A typical mating
scheme for this task is explained in Appendix 1.
A. Prokop - A rough guide to Drosophila mating schemes 11

Box 4. Gene descriptors and locators

 Drosophila genes have different descriptors: name, symbol, synonyms, the annotation symbol and
1
the FlyBase ID. As an example, go to the FlyBase home page flybase.org . In the "Quick Search" box
click on the "Data Type" tab, select "Data Class / genes" and type "shot" into the text field. This will
direct you to the gene page where you will find a full description of the gene short stop including
various identifiers and locators in the top section and further synonyms in the second last bottom
section [e.g. kakapo/kak, groovin/grv, kopupu/kop, l(2)CA4]. The naming of genes and chromosomal
aberrations follows clear rules (FlyBase / Documents / Nomenclature), and a few are summarised
here:
o The names of Drosophila genes (and their associated short forms or symbols) reflect the
classical (and certainly most human) way to describe a gene or marker mutation. They are
most commonly used in daily life and publications and tend to reflect the mutant phenotypes of
genes - often in very creative ways (e.g. faint sausage, ether-a-gogo, couch potato). For
example, white loss-of-function mutations cause white eyes, indicating that white gene function
is normally required for red eye colour. However, not everybody has followed this tradition when
naming genes. Furthermore, mutations of genes which were identified as homologues to known
mouse or human genes tend to be named after their mammalian relatives. Note that genes
encoding products of similar molecular function may be given names/symbol with identical
prefixes (usually indicating the protein class) and unique suffixes (usually referring to a gene's
cytogenetic location; e.g. Actin-5C, Actin-42A, Actin-57B). For an entertaining radio feature
about fly names listen to www.bbc.co.uk/programmes/b00lyfy1.
o As illustrated by the shot example, genes have often been called differently by independent
researchers (Synonyms & Secondary IDs), and these names come with their independent
symbols. FlyBase usually follows the rule that the first published name for a mutation of a gene
(usually not the wildtype locus or protein) becomes official, but a searchable list of all
synonymous names is maintained. In any case, FlyBase is your key point of reference and you
are advised to use their official naming.
o The annotation symbol (CG number, the Computed Gene identifier) originates from the
genome sequencing projects and has only been assigned to genetic loci that have been
identified as genes. For example, Cy/Curly is a mutation of unknown molecular nature and has
therefore no CG number. CG numbers are primarily used if no other name has been given yet.
o The FlyBase ID (FBgn = FlyBase gene) is the only unique identifier available for both
annotated genes and non-annotated marker mutations. It is often the prime reference during
database searches.
 As a general convention, genes/symbols that were FIRST named after recessive mutant alleles
(section 4.1.2) start with lower case, those FIRST identified by dominant alleles are capitalised. For
example, abd-A is lower case due to its original classification as a recessive gene, although
subsequent analyses have revealed dominant loss-of-function mutant phenotypes [8]. Capitalisation
can be confusing, since identical symbols starting with either upper or lower case represent different
gene or marker names (e.g. syn/syndrome versus Syn/Synapsin). Furthermore, genes named after
vertebrate homologues are capitalised, regardless of whether their mutant alleles are dominant or
recessive (e.g. Nrx-IV /Neurexin IV or Syn/Synapsin).
 Be aware that short hand for mutant alleles in daily use can differ. For example, "w;+;+" or "w" or "w-"
or "w-/w-" all mean the same thing, i.e. a white mutant fly. Whereas the first two versions do not
discriminate gender, the fourth option clearly indicates a female.
 Note that genes and their mutant alleles are usually italicised, whereas proteins are written in plain
and often capitalised (the shot gene, the shotsf20 mutant allele, the Shot protein)
 The genomic location of a gene is given in up to 4 ways: the chromosome (arm), cytogenetic map
position (both Fig. 5), the sequence location within the fully sequenced Drosophila genome and, for
marker mutations, also the recombination map position (e.g. the shot gene is on chromosome arm
2R, in cytogenetic map position 50C6-50C9, in sequence location 2R:9,751,742..9,829,615
corresponding to the recombination map position 2-[68]). Use the "Map Conversion Table"
(importable in Excel) for determining recombination map positions of other genes (section 4.1.4).

aaaaaaaaaaaaaaaaaa
1
for an easy guide to FlyBase see: http://flybase.org/static_pages/docs/pubs/FlyBase_workshop_2009.pdf
A. Prokop - A rough guide to Drosophila mating schemes 12

Figure 8. Anatomy of adult Drosophila

Lateral (A) and dorsal (A') view of the head and thorax region of an imago; body parts and bristles are
indicated. B) Ventral views of a female (left) and male (right) abdomen; note differences of the anal plate
in B which provide easy markers to determine gender (Fig. 4). C) Morphology of the wing and its
characteristic veins. Image modified from [45].
A. Prokop - A rough guide to Drosophila mating schemes 13

Figure 9. Examples of typical marker mutations used during genetic crosses

Mutations are grouped by body colour (top), eye markers (2nd row), wing markers (3rd row), bristle markers
(bottom row), and "other" markers (top right). Explanations in alphabetic order:
o Antennapedia73b (dominant; 3rd; antenna-to-leg transformation)
o Bar1 (dominant; 1st; kidney shaped eyes in heterozygosis, slit-shaped eyes in homo-/hemizygosis)
o Curly (dominant; 2nd; curled-up wings; phenotype can be weak at lower temperatures, such as 18ºC)
o Dichaete (dominant; 3rd; lack of alula, wings spread out)
o Drop (dominant; 3rd; small drop-shaped eyes)
o ebony (recessive; 3rd chromosome; dark body colour)
o Humeral (dominant; 3rd; Antennapedia allele, increased numbers of humeral bristles)
o Irregular Facets (dominant; 2nd; slit-shaped eyes)
o mini-white (dominant in white mutant background, recessive in wildtype background; any chromosome;
hypomorphic allele commonly used as marker on transposable elements)
o Pin (dominant; 2nd; short pointed bristles)
o Serrate (dominant; 3rd; serrated wing tips)
o singed (recessive; 1st; curled bristles)
o Stubble (dominant; 3rd; short, blunt bristles)
o vestigial (recessive; 2nd; reduced wings)
o white (recessive: 1st; white eye colour)
o yellow (recessive; 1st; yellowish body colour)
1
Photos of flies carrying these marker mutations have been published elsewhere [39,46] .

4.2. Marker mutations

The anatomy of the fly is highly reproducible with regard to features such as the sizes and positions
of bristles, the sizes and shapes of eyes, wings and halteres, or the patterns of wing veins (Fig. 8).
2
Many mutations have been isolated affecting these anatomical landmarks in specific ways [47] .
On the one hand these mutations can be used to study biological processes underlying body
patterning and development (by addressing what the mutant phenotypes reveals about the normal

1
or download the poster "Learning to Fly":
http://onlinelibrary.wiley.com/journal/10.1002/%28ISSN%291526-968X/homepage/free_posters.htm
2
available on FlyBase at the bottom of "Summary Information" for genes that were listed in the book
A. Prokop - A rough guide to Drosophila mating schemes 14

gene function). On the other hand these mutations provide important markers to be used during
genetic crosses and, hence, for mating scheme design. A few marker mutations commonly used for
fly work are illustrated in Fig. 9.

Figure 10. The use of balancers in stock maintenance

A cross of two parents (P) heterozygous for the homozygous embryonic lethal mutation lamininA (lanA)
and the recessive and viable marker mutation e (ebony, dark body colour). Both mutations are on the 3rd
chromosome and kept over a balancer. The mutant chromosome is shown in orange, the balancer
chromosome in red, parental alleles in blue, maternal in black. The first filial generation (F1) is shown on
the right. It is compared to a parallel cross (left) where the balancer was replaced by a wildtype
chromosome (white). In the parallel cross, only the two combinations containing lanA in homozygosis are
lethal (black strikethrough). Out of 6 viable combinations, only two are identical to the parents. In the
cross with balancers, also the homozygous balancer constellation is eliminated (blue strikethrough) as
well as all combinations involving recombination (red strikethrough). Only the combinations identical to
the parental genotype are viable, ideal for stock maintenance.

4.3. Balancer chromosomes

Balancer chromosomes are essential for the maintenance of mutant fly stocks as well as for mating
scheme design [3]. Balancer chromosomes carry multiple inversions through which the relative
positions of genes have been significantly rearranged. Balancer chromosomes segregate normally
during meiosis, but they suppress recombination with a normal sequence chromosome and the
products of any recombination that does occur are lethal due to duplications and deletions of
chromosome fragments (aneuploidy of chromosome fragments). In addition, most balancer
chromosomes are lethal in homozygosis. Together these properties are essential for stock
maintenance, since they eliminate all genotypes that differ from the parental combination (Fig. 10).
First chromosomal balancers (FM7, first multiply-inverted 7) are usually viable in homo- or
hemizygosis, but carry recessive mutations such as snX2 and lzs that cause female sterility in
homozygosis. The positive effect for stock maintenance is the same (Fig. 11). The third key feature
of balancer chromosomes is the presence of dominant and recessive marker mutations.
Through their dominant marker mutations balancer chromosomes are easy to follow in mating
schemes. For example, by making sure that a recessive mutant allele of interest is always kept
over dominantly marked balancers, the presence of this allele can be "negatively traced" over the
various generations of a mating scheme - especially since recombination with the balancer
A. Prokop - A rough guide to Drosophila mating schemes 15

chromosomes can be excluded. The following balancer chromosomes are commonly used (for
mentioned markers refer to Fig. 9; also see Tip 3):
a. FM7a (1st multiply-inverted 7a) - X chromosome
typical markers: y, wa, sn, B1
b. FM7c (1st multiply-marked 7c) – X chromosome
typical markers: y, sc, w, oc, ptg, B1
c. CyO (Curly derivative of Oster) - 2nd chromosome
typical markers: Cy (Curly), dp (dumpy; bumpy notum), pr (purple; eye colour), cn2 (cinnabar;
eye colour)
d. SM6a (2nd multiply-inverted 6a) – 2nd chromosome
typical markers: al, Cy, dp, cn, sp
e. TM3 (3rd multiply-inverted 3) - 3rd chromosome
typical markers: Sb, Ubx bx-34e, (bithorax; larger halteres) e, Ser
f. TM6B (3rd multiply-inverted 6B) - 3rd chromosome
frequent markers: AntpHu, e, Tb (Tubby; physically shortened 3rd instar larvae and pupae)
Note that the 4th chromosome does not require balancers since it does not display recombination.
Instead the ciD mutant allele is used to maintain stocks with lethal/sterile mutations of genes on the
4th chromosome; ciD is a recessive lethal, dominant marker mutation caused by a chromosome
rearrangement that led to a fusion protein encoded by the cubitus interruptus and pan genes.

Figure 11. First chromosome balancer, FM7

A stable stock carrying a recessive, homozygous lethal allele of myospheroid (mys) balanced over the
FM7 chromosome carrying the following marker mutations: recessive y (yellow body colour), recessive wa
(bright orange eyes), dominant Bar1 (reduced eyes; Fig. 6). In the F1 generation, hemizygous mys mutant
males die as embryos, females homozygous for FM7 are viable but sterile. Therefore, only the parental
genotypes contribute to subsequent generations, thus maintaining the mys mutant stock.

5. Transgenic flies
5.1. Generating transgenic fly lines
Transgenic flies have become a hub of Drosophila genetics with many important applications (see
below). Accordingly, transgenic animals are omnipresent in mating schemes, and it is important to
understand their principal nature and some of their applications. The generation of transgenic fly
lines is based on the use of transposable elements/transposons. Transposable elements are
virus-like DNA fragments that insert into the genome where they are replicated like endogenous
genes and can therefore be maintained in that position over many generations. Natural transposons
encode specialised enzymes called transposases. Transposases catalyse mobilisation of the
transposons into other genomic locations either through excision/re-integration or through
replication (Fig.12A). In Drosophila, the most frequently used class of transposon is the P-element
which will be mainly dealt with in this manual. For the purpose of transgenesis, transposons are
modified genetically. The transposase gene is removed and replaced by those genes the
A. Prokop - A rough guide to Drosophila mating schemes 16

experimenter wants to introduce into the fly genome. Furthermore, they contain marker genes and
genes/motifs for the selective cloning of the P-element in bacteria (Fig. 12B).
To introduce purpose-tailored transposons into the fly genome, they are injected into the
posterior pole of early embryos where they are incorporated into newly forming pole cells (Fig. 12)
[48]. Pole cells are the precursors of sperm and egg cells that will then give rise to a certain
percentage of transgenic offspring. To catalyse the insertion of these P-elements in the pole cell
genome, transposase-encoding helper elements are co-injected with them (or transgenic fly lines
are used that display targeted expression of transposase specifically in the germline). Helper
elements can themselves not insert/replicate and will gradually disappear when pole cells and their
progeny proliferate (Fig. 12D). Through this disappearance of the enzymatic activity, successful P-
element insertions are stabilised and can be maintained as stocks. Generating transgenic fly lines
through transposon/helper element injection requires technical expertise and specialised equipment
such as micromanipulators and glass needle pullers. It is often considered more economical to
outsource this task to specialised companies (of which there are a number existing worldwide),
instead of establishing and maintaining this capacity in individual laboratories.
Existing P-element insertions can be mobilised to produce excisions and transpositions into
new chromosomal locations. For this, a stock carrying the stable transposase-encoding insertion
P{∆2-3} is crossed with P-element-carrying flies to induce transposition. In the next generation,
P{∆2-3} is crossed out again to stabilise any newly generated P-element insertions [26]. P-element
mobilisation is used for a number of reasons. For example, random P-element insertions into genes
can disrupt their functions and provide new mutant alleles for these genes (P-element
mutagenesis) [26]. In other approaches, reporter genes on P-elements (e.g. lacZ, Gal4 or GFP)
are used to interrogate the genome for gene expression patterns (enhancer/gene/protein trap
screens; details in section 5.2.). Mobilisation of mapped P-element insertions can also be used to
induce deletions at their insertion sites. This can occur through a process called imprecise
excision where the P-element may remove genetic material either side of the insertion site.
Deletions can also be generated through homologous recombination, a strategy that removes the
genomic sequence between two adjacent P-element insertions [30]. For these latter approaches,
several transposable element insertions for most gene loci are readily available, which are carefully
listed in FlyBase and the Berkeley Drosophila Genome Project (BDGP) [49].

Tip 3. Special balancer chromosomes

Numerous balancer stocks with interesting features are available from Drosophila stock centres (e.g.
Bloomington / Balancers). A few are listed here:
 extra features on balancers can make your life easier:
o most 1st and 2nd chromosomal balancers carry the same dominant marker (B and Cy,
respectively); additional dominant markers, such as Star/S* on CyO or Lobe/L4 on SM1, can
be helpful to distinguish paternal and maternal balancers, e.g. in back-crosses
o balancers may carry l(2)DTS, a temperature-sensitive cell-lethal locus; by elevating the
temperature during development, the individuals carrying this balancer are automatically
eliminated, thus enriching for animals homozygous for the non-balancer chromosome
o green/blue balancers carry constructs expressing GFP or βGal, ideal to select against balanced
animals also in embryos, larvae or pupae - live or in fixed/stained preparations
 multiple-balancer stocks carry balancers on more than one chromosome, ideal to cross together
and keep mutations/markers on different chromosomes (see also Fig. 15)
 translocation balancer stocks also carry two balancers, but these act as one balancer across
different chromosomes; large fragments have been exchanged between these balancers [e.g.
T(2;3)CyO-TM3] causing lethality in animals that do not inherit both of them
 compound-X or attached-X chromosomes [e.g. C(1)DX] are not true balancers but can be used in
similar ways; they consist of two X chromosomes fused together so that they do not segregate during
meiosis and are jointly passed on to one gamete. Females are C(1)DX/Y because they inherit the
attached X from their mothers and a Y from their fathers; males are X/Y, i.e. only males pass on the
non-attached X chromosome, ideal for maintaining dominant female sterile mutations; C(1)DX/X
females are lethal or sterile.
A. Prokop - A rough guide to Drosophila mating schemes 17

Figure 12. Using P-elements to generate and map transgenic insertions

A) The insertion of natural P-elements into the genome (grey line) requires two key features: firstly,
flanking IS motifs (insertion sequences) mediating stem-loop conformation important for the insertion
process (blue arrow); secondly, catalytic activity of transposase (scissors and dashed blue arrow), an
enzyme encoded by the P-element itself. B) P{lacZ,w+} is a classic example of an engineered P-element
used for transgenesis where the transposase gene is replaced by: the lacZ gene of E. coli (dark blue
box), a mini-white gene as selection marker (see F; red box), an antibiotic resistance gene (e.g. to
ampicillin; white box) and an origin of replication (ori; grey box). Once a fly strain with a stable genomic P-
element insertion is established, the exact insertion site can be mapped: genomic DNA from these flies is
extracted and then digested using defined restriction sites in the P-element (e.g. EcoR1; red asterisk) and
random sites for the same enzyme (light blue box) in the nearby genome (grey letters); the obtained
restriction fragment contains the C-terminal part of the P-element permitting selective cloning along with
the adjacent genomic sequence; the obtained gene sequence can be blasted against the fly genome to
map its precise position (box with blue letters) and deduce the cytogenetic map position (box with green
letters; see Box 3). C-F) Making transgenic flies: a mix of P-elements and helper element (red) is injected
into the posterior pole of early embryos, where they become incorporated into the genome of pole cells
(C), the precursors of the gametes in the adult; the helper elements encode transposase which catalyses
the insertion of all genetic material flanked by IS motifs (D), but they lack IS motifs themselves, i.e. they
fail to insert and replicate but are diluted out during subsequent cell divisions; injected individuals mature
into w- adults that carry random P-element insertions in their gametes (E); after a cross to a w- stock, only
transgenic offspring display red eyes (due to the mini-white gene on the P-element) and can be selected
(F).
A. Prokop - A rough guide to Drosophila mating schemes 18

A number of problems with P-elements have been identified and led to improved strategies.
For example, P-elements have size limitations for the DNA inserts they can successfully insert into
the fly genome. Fragment sizes can be significantly increased through the use of BAC (Bacterial
Artificial Chromosome) technology, which allows whole genomic loci of greater than 100 kb to be
used for transgenesis [5,50]. Another problem is the so called position effect, which results in
identical P-element constructs having different levels of expression according to their genomic
insertion sites. Such position effects are due to the fact that each genomic locus displays its
individual transcriptional base-level and degree of chromosomal condensation. This problem can be
circumvented by using site-directed integration of transposons into known genomic positions. For
example, ΦC31 integrase (as an alternative to the P-transposase) promotes recombination
between attP and attB motifs. Consequently, when attB-bearing transposons are injected into
ΦC31-expressing fly strains carrying attP sites at defined genomic locations, a high percentage of
transposons will insert only at the defined attP site [51]. ΦC31-mediated recombination can also be
used to engineer genes or genomic regions within their natural chromosomal location (genomic
engineering) [9]. Finally, P-elements display a pronounced non-random insertion spectrum
(insertion hot & cold spots), meaning that certain classes of transposons are biased to insert in
certain regions of the genome avoiding others, or show preferential insertion in 5' regulatory rather
than coding regions of genes. This can be advantageous in some cases, but primarily poses a
problem in particular for genome-wide genetic screens (Fig. 2). To circumvent this problem and
complement existing P-element collections, a number of alternative vectors with different or less
pronounced preferences are available, such as the lepidopteran piggyBac or the Minos transposon
[26,49].

5.2 Important classes of P-element lines

There is a great variety of transgenic fly lines (Box 3) and their nomenclature is complex (see
FlyBase / Documents / Nomenclature). This nomenclature takes into consideration the respective
class of transposon, the molecular components it contains including dominant markers, the
insertion site and other unique identifiers. Here we use a "light" version of this nomenclature (Figs.
12 and 13), with P indicating P-element as the vector, information between curly brackets naming
the key transgenic components including w+ as the dominant marker, and further information
behind brackets may indicate the gene locus of insertion. Usually further identifiers in superscript
are required to unequivocally describe each individual insertion line but will not be considered here.
In the following some important classes of transgenic lines will be explained.
a. Enhancer/reporter construct lines (Fig. 13 A): In order to study regulatory non-coding regions
of genes, genomic fragments containing these regions can be cloned in front of a reporter
gene (e.g. lacZ from E. coli) fused to a P-element promoter which alone does not initiate gene
expression. Regulatory regions of genes contain enhancers, regulatory activators of gene
transcription which may act over distances of several kilo bases to facilitate transcriptional
initiation at gene promoters. Usually they act on promoters of endogenous genes in their
region, but also on P-element promoter of transgenic constructs. Transgenic fly strains with
these constructs can be used to analyse the spatiotemporal expression pattern of βGal (the
lacZ product), thus revealing tissue- or stage-specific enhancers regulating the transcription of
specific genes. Once lines with unique expression patterns have been generated, they may as
well be used as powerful genetic tools. For example, enhancer/reporter construct lines
carrying target sequences for a certain transcription factor may represent excellent reporters
reflecting the activity status of that specific transcription factor under experimental conditions.
b. Enhancer trap lines (Fig. 13 B): If a P-element carrying lacZ behind a P-element promoter is
inserted within the activity range of endogenous enhancers, lacZ expression can be induced
by these enhancers, often reflecting (aspects of) neighbouring genes' expression patterns.
This strategy has been used to systematically search for genes which are expressed (and
therefore potentially relevant) in specific tissues. This procedure is referred to as an enhancer
trap screen [52]. Since P-element insertions frequently affect the function of genes at their
insertion site (stippled red T in Fig. 13 B), they can be used for systematic P-element
A. Prokop - A rough guide to Drosophila mating schemes 19

mutagenesis screens (see Fig. 2) [26]. Once P-induced insertions have been generated,
lacZ staining patterns may reveal when and where the gene is active (Fig. 13 B), and efficient
cloning strategies can be used to map the insertion and identify the targeted gene (Fig. 12 B).
Transposon-based screens have been carried out with various technical modifications. For
example, protein trap screens select for insertions of specifically engineered transposons
into introns of genes (within or next to their coding regions). These transposons carry
sequences coding for protein tags (e.g. GFP) flanked by splice acceptor and donor sites.
During the natural splicing of the host gene, this tag sequence gets incorporated into the
splice product, thus fusing the tag to the endogenous protein. Many protein trap lines are
listed in FlyBase displaying fluorescent versions of endogenous proteins, allowing their natural
expression and localisation patterns to be studied [34,53].

Figure 13. Enhancer trap and enhancer/reporter lines

A) P{Ubx-lacZ,w+} illustrating an enhancer/reporter line. A transcription enhancer element that usually
activates the promoter of the Ubx gene at cytogenetic map position 89D (light green box with right
pointing arrow) is cloned (stippled black line) into a P-element; Ubx-E is cloned next to a lacZ reporter
gene with a basal promoter (dark box with right pointing arrow) that alone is insufficient to drive lacZ
expression. After genomic insertion (scissors; here at cytogenetic map position 36C), Ubx-E activates
(black arrow) transcription of the basal promoter in a Ubx-like pattern translating into a Ubx-like ßGal
expression pattern in the transgenic flies (blue). B) P{lacZ,w+}Ubx illustrating an enhancer trap line. A P-
element (curly bracket; colour code as in Fig. 12) carrying lacZ with a basal promoter is inserted in the
Ubx gene locus at 89D. The endogenous Ubx-E activates expression of the lacZ gene on the P-element
(blue in fly). Note that the inserted P-element may disrupt (red stippled T) expression or function of the
endogenous gene (red stippled X), thus generating a mutant allele (red stippled arrow).

c. Gal4/UAS lines: Gal4 is a transcription factor from yeast that activates genes downstream of
UAS (upstream activating sequence) enhancer elements. Gal4 does not exist endogenously
in flies and does not act on any endogenous loci in the fly genome. Very many transgenic
Gal4 fly lines have been and are still being generated. To illustrate this point, the simple
search term "Gal4" produces almost 6000 hits representing individual fly stocks at the
Bloomington Stock Centre alone. Of these, numerous Gal4 lines are readily available that
display Gal4 expression in different tissues or cells at specific developmental stages (Fig. 14
a, b). By simply crossing Gal4-expressing flies to UAS construct lines (Fig. 14 c, d) or
enhancer-promoter (EP) lines [54] (Fig. 14 e), the genes downstream of UAS enhancers are
being activated. UAS-linked genes can be of very different nature including reporters, different
isoforms of fly genes (or of other species), optogenetic or physiological tools, small interfering
RNAs or cytotoxins (Box 3). Once crossed to a Gal4 line, the offspring will display expression
of these UAS-coupled genes in the chosen Gal4 pattern. This provides an impressively
versatile and powerful system for experimentation, the spatiotemporal pattern of which can be
further refined through technical improvements such as the use of Gal80 (a Gal4 repressor),
dual binary systems or Split Gal4 [29].
A. Prokop - A rough guide to Drosophila mating schemes 20

Figure 14. The versatile Gal4/UAS system for targeted gene expression
The Gal4/UAS system is a two component system where flies carrying Gal4-expressing constructs are
crossed to flies carrying UAS-constructs (inset). Gal4 (black knotted line) binds and activates UAS
enhancers (dotted-stippled lines), so that the pattern in which Gal4 is expressed (here ubiquitously in the
fly) will determine the expression pattern of any genes downstream of the UAS enhancer (here ßGal or
Ubx). The two components can be freely combined providing a versatile system of targeted gene
expression. For example, Gal4-expressing constructs can be enhancer construct lines (a) or enhancer
trap lines (b). The shown Gal4 lines are analogous to those in Fig. 12 with some modifications: these P-
elements carry Gal4 instead of lacZ, the enhancer trap line is inserted into the ubiquitously expressed
Act42A actin gene at cytogenetic map position 42A, and the enhancer element is the Act42A enhancer
(actin-E) activating expression of Gal4 ubiquitously in the fly (black). Three examples of UAS lines are
shown: c) P{UAS-lacZ,w+} carries a UAS enhancer in front of the lacZ reporter gene; d) P{UAS-Ubx,w+}
carries the UAS enhancer in front of the Ubx gene; e) P{EP,w+}Ubx is an enhancer-promoter (EP) line
with a random insertion into the Ubx locus at 89D (analogous to enhancer trap line in Fig. 12 A). P-
elements of EP lines carry an UAS enhancer plus basal promoter which, on Gal4 binding, jointly activate
genes that lie downstream of their random insertion sites (here the Ubx gene).

d. FRT lines: FRT (FLP recognition target) sites are specifically targeted by the yeast FLP
recombinase ("flippase"). The FLP/FRT system is widely used in Drosophila as an inducible
recombination system that has mostly replaced former X-ray based strategies [31,55]. It is
used to excise genetic material (to activate/inactivate genes or markers) or to cause
somatic recombination between homologous chromosomes, an event that would normally
only occur during meiosis (Fig. 7). Somatic recombination requires specific insertions of FRT-
bearing P-elements close to the centromere of both homologous chromosomes. At these FRT
sites, FLP will catalyse breakage and exchange of the homologous chromosome arms which
can distribute into different cells in subsequent cell divisions. When starting from heterozygous
individuals, this method can produce mosaic tissues with homozygous clones of cells
surrounded by heterozygous cells [31]. Somatic recombination is used for MARCM (Mosaic
Analysis with a Repressible Cell Marker) analysis studying the behaviour of single mutant
cells or cell groups in normal or mutant tissue [56] (Fig. 15). Another important application is
the generation of germline clones in female gonads (Fig. 16). Germline clones are an
efficient strategy to generate maternally mutant embryos, i.e. to circumvent the problem of
maternal product [57]. Thus, mothers heterozygous for a homozygous lethal/sterile mutation
may deposit maternal product, consisting in mRNAs and/or proteins of their healthy gene
A. Prokop - A rough guide to Drosophila mating schemes 21

copy, in oocytes. Perdurance of his maternal product into embryonic or even postembryonic
stages may mask mutant phenotypes of homozygous mutant (zygotic mutant) offspring,
posing a problem for mutant analyses. Through using the antimorphic, dominant female sterile
ovoD1 or ovoD2 alleles, germline clone analyses positively select for successful recombination
events and have become highly efficient [57].

Figure 15. Clonal analysis using

MARCM:
A) MARCM scheme: Activation of
UAS-GFP through a tissue-specific
Gal4 driver (blue arrow) is suppressed
(grey T) by the Gal4 inhibitor Gal80
present in heterozygosis on a
chromosome with an FRT site; the
homologous chromosome carries a
mutation of interest and an equivalent
FRT site; activation of Flippase (Flp)
causes somatic recombination at these
FRT sites (red arrows); in a
subsequent mitosis and cell division
the mutant allele may assort into one
common daughter cell and become
homozygous (m/m) creating a parallel
twin clone (+/+) that carries both the
wildtype allele and Gal80 in
homozygosis. Subsequent divisions
multiply cells with these genotypes
embedded in heterozygous tissue. B-
D) Illustration for the use of MARCM in
research: B) Image of a normal wing
imaginal disc at the late larval stage
displaying blue marker gene
expression along the antero-posterior
compartment boundary. C) In larvae
carrying a particular mutation in homozygosis (m/m), wing discs express the marker gene throughout and
are under-grown and aberrant to a degree that no sensible conclusions can be made about the gene's
function. D) Small MARCM clones do not disturb the overall morphology of the wing disc and allow the
study of mutant cells unequivocally identifiable by their GFP-expression (green outline). In this example,
m/m mutant cells away from the compartment boundary display ectopic expression of the blue marker
gene, suggesting that the wildtype function of the gene behind m is to negatively regulate expression of
the marker gene.

e. RNAi lines: Application of RNA interference strategies in flies has become a powerful
alternative to the use of mutant alleles. As one key advantage, fly lines carrying UAS-RNAi
constructs (available for virtually every gene) [32] allow the targeted knock-down of specific
genes in a reproducible tissue or set of cells, often at distinct stages of development. Like
analyses using mutant clones (section 5.2d), this approach can therefore overcome problems
caused by systemic loss of gene function, such as early lethality (often impeding analyses at
postembryonic stages) or complex aberrations of whole tissues that can be difficult to
interpret. However, the use of RNAi lines needs to be well controlled. Demonstration of
reduced protein or RNA levels of the targeted gene is not sufficient, since phenotypes can still
be due to addtitional off-target effects (i.e. knock-down of independent gene functions).
Therefore, it is advised to use more than one independent RNAi line targeting different regions
of the gene. Other proof of specificity can come from enhancement of the knock-down
phenotype in the presence of one mutant copy of the targeted gene or, vice versa,
suppression of the knock-down phenotype through co-expression of a rescue construct for the
targeted gene (ideally carrying a mutation that does not affect its function but makes it
immune to the knock-down construct).
A. Prokop - A rough guide to Drosophila mating schemes 22

Figure 16. Maternal gene product and germline clones

A) Many genes are expressed in the female germline and gene product in the form of mRNA or protein
(blue) is deposited in oocytes but not in sperm (white), often perduring into early embryonic stages or
even larval stages and beyond (depending on the half life of the particular mRNA and/or protein). B) If
mutant alleles of such genes are homozygous lethal, heterozygous parents (m/+) have to be used in
crosses to generate homozygous mutant F1 individuals (m/m); these mutant individuals display maternal
gene product (derived from their mother's wildtype copy of the gene) that may mask mutant phenotypes
especially at earlier stages of development. C) Heterozygosis for ovoD causes cell-autonomous
elimination of female germline cells; through FRT-mediated recombination in m/ovoD mothers,
homozygous oocytes can be generated that are no longer eliminated and are the only eggs being laid by
these females [57]. Homozygous mutant embryos developing from these eggs lack both maternal
function and zygotic function, whereas heterozygous embryos display zygotic expression of the wildtype
allele starting earliest at ~3.5 hr after fertilisation at 25ºC (embryonic stage 8) [58].

6. Classical strategies for the mapping of mutant alleles or transgenic constructs

You may encounter situations in which the location of a mutant allele or P-element insertion is not
known, for example after having conducted a chemical or X-ray mutagenesis (Fig. 2) or when using
a P-element line of unknown origin (unfortunately not a rare experience). To map such mutant
alleles, a step-wise strategy can be applied to determine the chromosome, the region on the
chromosome and, eventually, the actual gene locus. Nowadays, mapping can often be achieved by
molecular strategies, such as plasmid rescue (Fig. 12 B), inverse or splinkerette PCR [59] or high-
throughput genome sequencing [60]. However, classical genetic strategies remain important and
are briefly summarised here.
a. Determining the chromosome: You hold a viable P{lacZ,w+} line in the laboratory that serves
as an excellent reporter for your tissue of interest, but it is not known on which chromosome
the P-element is inserted. To determine the chromosome of insertion you can use a simple
two-generation cross using a w- mutant double-balancer stock (Fig. 17).
b. Meiotic mapping: During meiosis, recombination occurs between homologous chromosomes
and the frequency of recombination between two loci on the same chromosome provides a
measure of their distance apart (section 4.1.4). To make efficient use of this strategy, multi
marker chromosomes have been generated that carry four or more marker mutations on
the same chromosome (Bloomington / Mapping stocks / Meiotic mapping). Each marker
provides an independent reference point, and they can be assessed jointly in the same set
of crosses, thus informing you about the approximate location of your mutation [2,25]. Note
that multi-marker chromosomes can also be used to generate recombinant chromosomes
where other strategies might fail. For example, recombining a mutation onto a chromosome
that already carries two or more mutations, or making recombinant chromosomes with
homozygous viable mutations is made far easier with multi-marker chromosomes.
c. Deletion mapping: Deficiencies are chromosomal aberrations in which genomic regions
containing one, few or many genetic loci are deleted. Large collections of balanced
deficiencies are available through stock centres (e.g. Bloomington / Deficiencies) and listed
A. Prokop - A rough guide to Drosophila mating schemes 23

in FlyBase. Using improved technology the Bloomington Deficiency Kit now covers 98.4% of
the euchromatic genome [61]. These deficiencies provide a rich resource to map genes
through classical complementation testing. For this, you cross your mutant to deficiencies of
the region determined by meiotic mapping. If your mutation crossed to the deficiency
displays its known phenotype (e.g. lethality) you can infer that the gene of interest is
uncovered by this deficiency (hemizygous constellation). Note that, when dealing with
lethal mutations, only 25% of your offspring are expected to carry the phenotype, so you
look for presence/absence of balancer-free animals in F1 (Fig. 6). Absence of the phenotype
excludes the group of genes uncovered by the deficiency. By using various deficiencies in
the area, the mapping of the gene can be further refined (Fig. 18).

Figure 17. Determining the chromosome of insertion of a P-element

A homozygous viable transgenic fly line carries a P{lacZ,w+} insertion on either 1st, 2nd or 3rd chromosome
(Pw+?). P) To determine the chromosome of insertion, males of this line (paternal chromosomes in blue)
are crossed to a white mutant double-balancer line carrying balancers on both 2nd and 3rd chromosome
(note, that the same can be done in two parallel crosses to single balancer stocks carrying balancers on
only 2nd and only 3rd; try it out!). F1) In the first filial generation potential X chromosome insertions can be
determined; if X is excluded, complementary chromosome combinations are selected for a second cross;
make sure that males are used for the dominant marker combination (If and Ser) to prevent unwanted
recombination (section 4.1.4.), whereas recombination in the females is excluded by the balancers (CyO
and TM3). F2) In the second filial generation, potential 2nd or 3rd chromosomal insertions can be
determined; note that helpful stocks for follow-up crosses can be selected at this stage (e.g.
If/CyO;Pw+/Pw+ would facilitate future combinations of the P-element insertion with a mutation on the 2nd
chromosome); if w/w;If/CyO;Ser/TM3,Sb flies in F2 are still orange, you have a rare event in which your
insertion is on the 4th or the Y chromosome.
A. Prokop - A rough guide to Drosophila mating schemes 24

Figure 18. Deletion mapping

A mutation (red triangle) in the yellow highlighted gene locus is roughly mapped to a region of the right
arm of chromosome 2 (2R). To refine its mapping, the mutant allele is crossed to deficiencies (Df) that
have their breakpoints in this region (red bars indicate the deleted chromosomal region for each
deficiency). Closest breakpoints of deficiencies that complement the mutation (+) indicate the region in
which the gene is located (blue double-arrow). Closest breakpoints of non-complementing deficiencies (-)
may lie within the gene in question and, in this example, clearly identify the mutated gene (red double-
arrow).

d. Complementation tests with known loss-of-function mutant alleles: Once the location of your
gene has been narrowed down by deletion mapping, you can cross your mutation to
available loss-of-function mutations for the genes in this area, basically following the same
strategy as for deletion mapping. Presence of the phenotype indicates that the mutations
are alleles of the same gene (hetero-allelic constellation). Absence of the phenotype
suggests that these alleles belong to different genes (trans-heterozygous constellation).
However, be aware that the nature of a gene may be complex and lead to false
interpretations of your complementation analysis:
 Genes may display transvection, a phenomenon where different homozygous mutant
alleles affecting different areas of the same gene may complement each other [62].
 Genes can be nested, i.e. complete genes can be lying within introns of another gene,
or they may map to the complementary strand of DNA at the same locus.
 Coding regions of genes may be separate, but they may share the same enhancers.
 Finally, non-coding RNAs are encoded by independent loci that may often be
considered to represent genes themselves. These loci have important gene regulatory
1
functions and can complicate the analyses of other genes in their vicinity .
To circumvent some of these problems, other strategies are available. For example,
collections of UAS-RNAi fly lines (section 5.2e) can be used to systematically knock down
the functions of genes in the area of interest. This strategy only works if your mutation has
phenotypes characteristic enough to be unequivocally identifiable upon gene knock-down.
Furthermore, important clarification can often be obtained from the detailed transcriptional
profiles displayed for every gene on FlyBase (at the bottom of the "Expression/Regulation"
view in GBrowse).

7. Concluding remarks
You should now have gained the key knowledge and terminology required to design mating
schemes for Drosophila and to function in a fly laboratory. However, the information given is still

1
nice example: http://biobabel.wordpress.com/2012/05/30/a-dual-purpose-rna-and-hox-regulation/
A. Prokop - A rough guide to Drosophila mating schemes 25

basic and requires that you further explore the details behind the various aspects mentioned here.
For this, some literature has been provided throughout the text. Should there be mistakes,
passages that are hard to understand or information that is missing or wrong, please, be so kind to
let me know ([email protected]).

Box 5. How to design mating schemes (illustrated in Figs. 6 and 17)

 write 'X' between two genotypes to indicate the crossing step
 genes on the same chromosome may be separated by comma, and also the names of balancer
chromosomes may be separated by comma from the list of their markers (e.g. TM3,Sb,e)
 genes on homologous/sister chromosomes are separated by a slash or horizontal lines (usually one,
sometimes two)
 genes on different chromosomes are separated by a semicolon
 always write chromosomes in their order (1st ; 2nd ; 3rd); to avoid confusion indicate wildtype
chromosomes as "+" (e.g. y/Y ; + ; Sb/+); note, that the 4th chromosome is mentioned only in the
relatively rare occasions that 4th chromosomal loci are involved in the cross
 the first chromosome represents the sex chromosome; always assign a Y chromosome to the male of
a cross (see Fig. 6); note that the Y chromosome is sometimes indicated by a horizontal line with a
check on its right side ( )
 especially as a beginner, stick to a routine order, such as...
o ...the female genotype is always shown on the left side, male on right
o ...the maternal chromosomes (inherited from mother) are shown above, paternal chromosomes
(grey) below the separating line
 especially as a beginner, always write down all possible combinations resulting from a cross; carefully
assign phenotypes to each genotype, define selection criteria and check whether these criteria
unequivocally identify the genotype you are after
 to keep this task manageable, use curly brackets for chromosome separation and assess each
chromosome individually (Fig. 6). At the end, cross-check whether criteria might clash (for example, a
mini-white marker on the second chromosome only works as a selection criterion if the first
chromosome is homo- or hemizygous for white)
 always make sure that you avoid unwanted recombination events by using balancer chromosomes
and/or the recombination rules (no crossing-over in males or on the 4th chromosome). If
recombination is the task of your cross, make sure you use females during the crossing-over step
(usually in F1).
 be aware of fly nomenclature which can be confusing, especially with respect to capitalisation and the
indication of whether an allele is recessive, dominant, loss- or gain-of-function (Box 3). Be aware that
you understand the nature of the involved alleles, since dominant alleles behave differently to
recessive ones in a cross (Fig. 6)
 The nomenclature of transposable elements or chromosomal aberrations can be tedious. To work
more efficiently, feel free to use your own unequivocal short hand during the task. For example,
"P{UAS-lacZ,w+}" and "P{eve-Gal4,w+}" could be shortened to "PUw+" and "PGw+".

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A. Prokop - A rough guide to Drosophila mating schemes 28

Appendix 1. A recombination scheme

You want to recombine mutant alleles of the viable, recessive, 3rd chromosomal loci rosy (ry; dark
brown eyes) and ebony (e; black body colour) onto one chromosome. According to FlyBase, ry
localises to recombination map position 3-52, and e to 3-70.7. Hence, they lie 18.7cM apart,
indicating that statistically slightly less than 1 in 5 oocytes will carry the desired recombination
event.

For this, you start by crossing ry females with e males or vice versa (P, parental cross). In the first
filial generation (F1), all flies are trans-heterozygous (ry,+/+,e). Note that the different fly stocks
used in this cross will be colour-coded to allow you to easily trace the origin of each chromosome.

According to the recombination rule, you need to take females so that recombination can occur.
Note that crossing-over during oogenesis in these females occurs at random, i.e. their eggs which
give rise to the second filial generation (F2) represent a cocktail of recombination events with a
statistical likelihood of 18.7% as calculated above. Note that only half of the tested animals carry
the first marker ry, out of which only 18.7% display the wanted recombination. Therefore, 9.35% of
the single F2 individuals carry a recombinant chromosome with both markers, and 9.35% a
recombinant chromosome with wildtype alleles of both markers. The key task is to identify and
isolate these recombination events through a step-wise process.

In the first step, recombination events need to be "stabilised" to prevent further recombination. For
this, F1 females are crossed to a balancer stock carrying a balancer chromosome (Bal1) over a
dominantly marked chromosome (M1; sections 4.2. and 4.3). In the third filial generation (F3), you
A. Prokop - A rough guide to Drosophila mating schemes 29

determine whether one of the markers (here ry) is present (remember that, according to the law of
segregation, only 50% of balanced F2 individuals carry ry). To determine the presence of ry, you
cross F2 animals back to a ry mutant stock. Two important issues need to be considered here.
 Firstly, each individual in F2 is the result of an individual recombination event in its mother's
germline. Therefore, single animals need to be tested for the presence of ry. For practical
reasons, use single males since they can fertilise several females and therefore have a higher
likelihood to generate enough offspring.
 Secondly, you have to cross back to ry mutant flies, but need to be able to distinguish your
recombinant chromosome from the ry chromosome of the back-cross. For this, cross the ry
stock to a balancer stock (Bal2) that can be distinguished from Bal1.
In F3, use simple selection to separate out two groups of flies: non-balanced flies allow you to
determine whether flies have brownish eyes (i.e. carry ry on their potentially recombinant
chromosome). If this is the case, flies carrying Bal2 over the potentially recombinant paternal
chromosome (rather than the ry chromosome of their mothers) can be used to establish a stable fly
stock. The fourth filial generation (F4) emerging from these newly established fly stocks will contain
non-balanced animals (ry and e are viable mutations). Stocks in which non-balanced flies have
brownish eyes and dark body colour bear the desired recombinant chromosome and will be kept,
the rest discarded.

Tips: To have a statistical chance of isolating recombination events, more than 10 single crosses in
F2 should be used to match the 9.35% chance of obtaining a recombinant. Furthermore, the
example of ry and e represents an unusual case, since they are common marker mutations that are
found on several balancer chromosomes (section 4.3.). Using balancers with these markers would
allow you to immediately identify the presence of the desired mutations on the potentially
recombinant chromosomes. Try it yourself.
A. Prokop - A rough guide to Drosophila mating schemes 30

Appendix 2. A trihybrid cross

Example of a trihybrid cross between heterozygous parents (P, top) involving recessive alleles on X, 2nd and
3rd chromosomes (separated by semicolons). Homologous alleles are separated by a horizontal line; maternal
alleles are shown in black, paternal ones in blue. Mutant alleles are w (white; white eyes), vg (vestigial;
reduced wings), e (ebony; dark body colour); phenotypes are indicated by fly diagrams (compare Fig. 9). In
the first offspring/filial generation (F1) each chromosome has undergone independent assortment of alleles
(demarcated by curly brackets) and each of the four possible outcomes per chromosome can be combined
with any of the outcomes of the other two chromosomes resulting in 4 x 4 x 4 = 64 combinations. The Punnett
square at the bottom systematically lists all possible combinations (different phenotype classes are colour-
coded and display a 18:18:6:6:6:6:2:2 distribution; symbols are explained at the bottom). Red and blue
stippled boxes show the same examples of two possible offspring in both the curly bracket scheme and the
Punnett square. Note that the Punnett square reflects the numerical outcome of this cross in its full
complexity, whereas the curly bracket strategy only qualitatively reflects potential combinations and is easier
to interpret for the purpose of mating scheme design (Box 5).