Transgenic animals

M. Sc. Biotechnology
(IV Semester: Advanced topics)
 Terminologies
• Syngeneic: Identical (Inbred)
• Congenic: genetically identical except at a
single genetic locus or region (To study gene of
interest)
• Adoptive cell transfer method: Recipient
irradiation
 Transgenic
• Transfer of a foreign gene into the genome of
host through recombinant DNA technologies
• Applications
– Scientific Research
– Commercial Purposes (Increase in desired
properties: growth rate, disease resistance,
nutritional value, etc.)
Transgenics Development for Research
• Answer specific research question
– Eg: To study the role of specific protein in a
pathway
– KO, KI, constitutive or inducible modulation of
protein of interest, tissue specific expression
studies
– Development of animal model of diseases
 Steps Involved
• Obtain gene of interest
• Obtain appropriate host cells
• Introduction of gene of interest in host cell
• Integration in host genome
• Selection of recombinant cells
• Putting recombinant cells back in to host
• Check expression of protein of interest linked
to gene in produced progeny
 Desirable Host cells
• Desirable properties in host cells
– Good transfection rate and
in vitro survival
superovulation
– Faster cell division
– Better recombination frequency
– Good re-engraftment in the host
• Early stage embryo
(Fertilized egg with pronuclei,
Morula)
• Embryonic stem cells
 Introduction of gene of interest
• Microinjection
• Engineered embryonic stem cells
• Advanced recombination systems (Cre-LoxP)
• Retroviral vectors
• Sperm mediated transfer during fertilization
 Microinjection
• Most commonly used method
• Dissecting microscope + two micropipettes
(holding, injection)
• Volume of Foreign DNA-picoliters
• Linear copies of gene, bacterial plasmids
have better efficacy
• Mainly transferred in male pronuclei
• Microinjected embryo are cultured in vitro
up to morula, blastocyst stage before implant
• Need to check mouse pups at DNA, RNA, and
protein levels
• Expression will vary in transgenic offspring:
due to position effect and copy number
Efficiency of Microinjection method
 Transfer through Embryonic Stem cells
• More targeted as compared to DNA
microinjection due to use of selection markers
post gene insertion
• ES are maintained on feeder layer (fibroblast)
in-vitro during the process
• Successful in mice mainly (lack of in vitro
culture stability in other mammals)
Generation & selection of desired ES cells: For KO mice

(Necessary for ganciclovir action)

Retroviral Vector Linker Based
based gene Sperm-Mediated
Gene Transfer (LB-
transfer
SMGT)
 Cre-LoxP Technology
• Cre-lox is an incredibly popular and powerful site
specific recombinase (SSR) system
• Cre-lox technology was introduced in the 1980s
(Sauer and Henderson 1988; Sternberg and Hamilton
1981) and patented by DuPont Pharmaceuticals.
• Bacteriophage P1 has a cyclization recombination
called cre and particular DNA sequences called lox P
sites. The lox P sites work in pairs and they flank a
segment of DNA [target].
• Two have to be artificially introduced into the model
organisms
• LoxP sites:
• Cre mice can be designed to express Cre
recombinase.
• a loxP mouse contains two loxP sites that flank a
genomic segment of interest, the "floxed" locus
• Usually, Cre and loxP strains are developed
independently and then crossed.
• Different Cre strains, each containing a
Cre transgene under the direction of a different
tissue-specific promoter, may be crossed with a
single loxP strain
• a variety of Cre-mediated model systems can be
constructed, including transgenics, knockouts,
hypomorphs, repairable hypomorphs,
chromosome aberrants and diet-induced mutants.
 Components of Cre-LoxP system
• Cre expressing strains: Expresses the Cre transgene that is
under the control of a general or tissue-specific (conditional)
promoter. E.g. lck promoter (specific to T-cells), cytokeratin
promoter (specific to epithelial cells) etc. [general or
conditional knockouts].
• Inducible Cre strains: expresses modified Cre transgene that is
non-functional until an inducing agent (such as doxycycline,
tetracycline, type I-interferon, or tamoxifen) is administered at
a desired time point in embryonic development or adult life
• LoxP-flanked (floxed) strains: contains LoxP sites flanking (on
each side of) a critical portion of a target gene
• Cre reporter strains: contains LoxP sites along with reporter
(fluorescent or lacZ) marker proteins so as to trace success of
Cre recombination and/or alterations in gene expression.
Cre-LoxP recombination outcomes
 Applications
• Global Gene Deletion in Every Cell in the Mouse: the cre-recombinase
produced under a global promoter (present in all cells, e.g. EIIa or
Sox2). homozygous cre-flox strain will result in global deletion of gene
of interest
• Cell-, Tissue- or Organ-Specific Gene Deletion in the Mouse: expresses
cre-recombinase under a cell-, tissue- or organ-specific promoter. Eg:
Alb-cre mouse to study deletion in the liver
• Delete a Gene at a Particular Time Point With an Induction Stimulus: a
complete gene knockout mouse could be lethal if the gene deleted is
essential for embryonic development or other crucial cell functions. In
such cases, an induced Cre-loxP system is ideal.
• Tracking of cells: Putting a fluorescent protein after your gene of
interest and watch the turning on or off of the gene with cre
recombination.
• For induction of endogenous or transgenic
promoters, two main systems:
-Tetracycline; Tet-On and Tet-Off
-Tamoxifen; ER/4-OHT

spatiotemporally control
 Advantages: Cre-LoxP
• Contains DNA that is not found in animals or plants no
undesired activity
• Works in almost any type of cell, specific tissue, whole
organism.
• Wide application such as, labeling specific cell type in tissue,
differentiating particular cell.
• A more powerful approach to controlling gene expression has
also been developed by combining the Cre/loxand the
tetracycline-regulated transcriptional systems.
Disadvantages: Cre-LoxP
 Disadvantages: Cre-LoxP

• Not all tissue specific promoters are perfectly

specific. Basal levels of expression in other cell
types can sometimes cause unintended gene
expression.
• Requires a significant amount of time and
money.
 FLP-FRT system
• FLP-FRT system is similar to the Cre-lox system
• more frequently used in mouse-based
research
• flippase (FLP) recombinase, derived from the
yeast Saccharomyces cerevisiae.
• FLP recognizes a pair of FLP recombinase
target (FRT) sequences that flank a genomic
region of interest
 FLP-FRT system
• The first version of FLP discovered has a temperature
optimum of 30 °C and is therefore inefficient in
mammalian cells. Smart molecular evolution led to the
identification of FLPe which has a temperature optimum
of 37 °C.
• FLPe’s performance was further improved, by adding a
nuclear localization sequence (SV40 Large T nuclear
localization sequence) and expressing it from a CAGGs
promoter.
• However, FLPe is still less efficient than Cre recombinase.
• Differences in recombinase efficiency are important to
keep in mind when deciding whether to use the Cre-lox or
 Next ???
• PhiC31 recombination system
• zinc finger nuclease-assisted genetic
manipulations
Relative simplicity, accessibility to researchers
and the availability of a number of pre-existing
lox and FRT-carrying PSC lines, makes the Cre
and Flp systems a practical method of choice
for the site-directed integration of transgenes.
•PhiC31 integrase mediates the
integration of a 35-nucleotide AttB
site with a 35-nucleotide AttP site
to form an AttL and an AttR site.
•Because the AttL and AttR sites
are not targets of phiC31, this
reaction is irreversible (unlike
comparable reactions with cre and
flp).
•Once the barcodes are joined,
they can be amplified by PCR
(using primers complementary to
the arrows) for sequencing.
 SCID-Immune reconstitution

Mice homozygous for the severe

combined immune deficiency
spontaneous mutation Prkdcscid,
commonly referred to as scid, are
characterized by an absence of
functional T cells and B cells,
lymphopenia,
hypogammaglobulinemia, and a
normal hematopoietic
microenvironment.
Prkdcscid mice accept allogeneic and
xenogeneic grafts making them an
ideal model for cell transfer
experiments.
In general, Prkdcscid leakiness is low on
the NOD/ShiLtSz genetic background.
 RAG KO

Mice homozygous for the Rag1tm1Mom mutation produce no mature T cells or B cells.
Whereas Prkdcscid homozygotes produce some B cells and IgM (i.e., are
"leaky"), Rag1tm1Mom homozygotes lack all mature lymphocytes (i.e., are "non-
leaky"). Rag1 null mice have no CD3+ or T cell receptor (TCR) alpha-beta positive
cells. The thymus of the mutant mice contains 15 to 130 times fewer cells than
heterozygous or wild-type siblings. The thymocytes are CD8-CD4- and most are IL2
receptor-positive. Neither the spleen nor the bone marrow contain any IgM or IgD
staining cells, indicating an absence of mature B cells. These and other data suggest
that B cell and T cell development has been arrested at an early stage.
Macroscopically, the mutants are indistinguishable from heterozygotes or normal
wild-type siblings.
Autoimmunity Disease Model
MRL/lpr
This strain is commonly known as MRL-lpr or lpr mutant. Mice are homozygous for
the lymphoproliferation spontaneous mutation (Faslpr), and show systemic
autoimmunity, massive lymphadenopathy associated with proliferation of aberrant T
cells, arthritis, and immune complex glomerulonephrosis. Mice are useful as a model
to determine the etiology of systemic lupus erythematosus (SLE) and Sjorgren (Sicca)
syndrome and to evaluate therapies.
Date
Transgenic animals: timeline of key events
Event People Places
23 Jun 1925 Oliver Smithies was born in Halifax, United Kingdom Smithes University of Washington, University of North
Carolina
1929 Jackson Memorial Laboratories established to develop Jackson Memorial Laboratoroies
inbred strains of mice to study the genetics of cancer
and other diseases
19 Aug 1929 Frank Ruddle was born in West New York, New Jersey Ruddle Yale University

1974 First publication on inserting foreign DNA into mice Jaenisch, Mintz Salk Institute, Fox Chase Institute for Cancer
Research
September 1980 Scientists reported the first successful development of Barbosa, Gordon, Yale University
transgenic mice Plotkin, Ruddle, Scangos
November 1980 Technique published using fine glass micropipettes to Capecchi University of Utah
inject DNA directly into the nuclei of cultured
mammalian cells. High efficiency of the method enables
investigators to generate transgenic mice containing
random insertions of exogenous DNA.

5 Nov 1981 First successful transmission of foreign DNA into Constantini, Lacy Oxford University, Yale University
laboratory mice
December 1982 Giant mice made with the injection of rat growth Brinster, Palmiter University of Pennsylvania, University of
hormone Washington Seattle
1983 Course started in the molecular embyology of mice Costantini, Hogan, Lacy Cold Spring Harbour Laboratory, NIMR, Sloan
Kettering Cancer Research Center, Columbia
University
1985 First transgenic mice created with with genes coding for Kohler, Rusconi Max-Planck Institute
both the heavy and light chain domains in an antibody.

6 Nov 1987 Publication of gene targeting technique for targetting Thomas, Capecchi University of Utah
mutations in any gene
1988 Patent application filed for a method to create Bruggeman, Caskey, Laboratory of Molecular Biology, Babraham
transgenic mice for the production of human antibodies Neuberger, Surani, Teale, Institute, Cambridge University
Waldmann, Williams
12 Apr 1988 OncoMouse patent granted Leder, Stewart Harvard University
12 Jun 1992 First transgenic mouse model created for studying link Li, Bestor, Jaenisch Whitehead Institute for Biomedical Research
between DNA methylation and disease
1994 First transgenic mice strains reported for producing Bruggemann, S.Green, Cell Genesys, GenPharm, Laboratory of
human monoclonal antibodies Lonsberg, Neuberger Molecular Biology
Date Event People Places
January 1996 Dolly the sheep was cloned by Professor Ian Wilmut Roslin Institute
Wilmut's team at the Roslin Institute in
Edinburgh
5 Jul 1996 Dolly the sheep, the first cloned mammal, Wilmut, Campbell Roslin Institute
was born
14 Feb 2003 Dolly the sheep, the first cloned mammal died Wilmut Roslin Institute
September First fully human monoclonal antibody drug Agensys, Amgen
2006 approved
2007 Nobel Prize for Physiology for Medicine Capecchi, Evans, University of North Carolina,
awarded for discoveries enabling germline Smithies University of Utah
gene modification in mice using embryonic
stem cells
10 Mar 2013 Frank Ruddle died in New Haven, Connecticut Ruddle Yale University
26 Oct 2013 Michael Neuberger died Neuberger Laboratory of Molecular Biology
23 Sep 2015 Beijing Genomics Institute announced the Beijing Genomics Institute
sale of the first micropigs created with the
help of the TALENs gene-editing technique
5 Oct 2015 CRISPR/Cas9 modified 60 genes in pig Church Harvard University
embryos in first step to create organs suitable
for human transplants
10 Jan 2017 Oliver Smithies died Smithies University of Washington, University
of North Carolina
20 Apr 2017 Diabetes research using transgenic mice Menzies University of Edinburgh, University
shows the protein P2X7R plays important role College London, Imperial College
in inflammation and immune system offering
new avenue for treating kidney disease
23 Jan 2019 CRISPR-Cas9 used to control genetic Grunwald, Gntz, University of California San Diego
inheritance in mice Poplawski, Xu, Bier,
Cooper
Examples of Transgenic
Animals
(commercial applications
after ethical & safety
aprovals)
 Transgenic
Cattle
• Dairy cows carrying extra copies of two types of
casein genes produce 13% more milk protein
• Not only will this make the milk more nutritious, it
would allow for less milk to make more cheese
• Currently the milk from these animals is under FDA
review
• The important difference between this & other
transgenics is that the DNA added is not foreign
 EnviroPig TM
• Pig waste is a major pollutant & can cause eutrophication of lakes & streams
• Normally the phytic acid/phosphorus complex passes through the pig and is excreted
as waste
• Transgenic pigs express phytase in their salivary glands
• Phytic acid in the pig meal is degraded releasing phosphorus
• The phosphorus is absorbed by the pig

Transgenic Fish
• Tilapia, Salmon/trout, Catfish
• Can grow up to 6 times faster than wildtype fish
• Most have extra copies of growth hormone (GH) gene
• USFDA Approved GE Salmon for consumption in Nov, 2015
Animal Bioreactors “Pharming”
1) First transgenic sheep developed to produce a recombinant
protein drug in milk
• alpha-1-antitrypsin (AAT) treatment for emphysema & COPD
2) Transfer of the silk gene from Orb spiders into goats
• Each goat produces several grams of silk protein in her milk
• The silk is extracted, dried to a white powder, and spun into
fibers
• The fibers are stronger and more flexible
3) GTC Biotherapeutics has received approval to sell
human anti-thrombin (ATryn) purified from goat’s
milk in Europe
 Agriculture
• to develop new and improved strains of
livestock
• enhanced prolificacy and reproductive
performance, increased feed utilization and
growth rate, improved carcass composition,
improved milk production and/or
composition, modification of hair or fiber, and
increased disease resistance.
 Enhanced Nutrition
• improvement of nutrients in animal products,
including their quantity, the quality of the whole
food, and specific nutritional composition
• enhancing the omega-3 fatty acid in fish
consumed by humans may contribute to a
decreased occurrence of coronary heart disease
• transgenic pigs that contain elevated levels of
omega-3 fatty acids have been produced;
enhance the nutritional quality of pork
 Reduced Environmental Impact
• Pigs do not fully utilize dietary phosphorus.
• Dietary supplementation results in increased production
costs, and incomplete utilization results in phosphorus
levels in waste products that can cause pollution
problems.
• Golovan et al. (2001) reported the production of
transgenic pigs expressing salivary phytase as early as 7
days of age.
• The salivary phytase provided essentially complete
digestion of dietary phytate phosphorus in addition to
reducing phosphorus output by up to 75%.
 Diary Industry
• produce a greater quantity of milk
• produce milk of higher nutrient content
• produce milk that contain beneficial ‘nutriceutical' protein
• Over expression of beneficial proteins in milk through the use of
transgenic animals may improve growth, development, health, and
survivability of the developing offspring.
• Some factors that have been suggested to have important biological
functions in the neonate that are obtained through milk
include IGF-I, EGF, TGF-β, and lactoferrin
• altering the metabolism or uptake of cholesterol and/or fatty acids,
the content of fat and cholesterol of meats, eggs, and cheeses could
be lowered.
• receptors such as the low-density lipoprotein (LDL) receptor gene
and hormones like leptin are potential targets that would decrease
fat and cholesterol in animal products
 Mammary expression of
transgenic proteins
Species Where Promoter Reference
Protein Expressed
Expressed

Bovine αs1-casein (Maga et al. 2006)

Lysozyme goat

Lysostaphin Ovine β-lactoglobulin (Wall et al. 2005)

cattle

Bovine β and κ casein Bovine β-casein (Brophy et al. 2003)

cattle

IGF-I Bovine α-lactalbumin (Donovan et al. 2001)

pig

α-lactalbumin (Bleck et al. 1998)

pig Bovine α-lactalbumin

IGF-I Bovine αs1-casein (Wolf et al. 1997)

rabbits

(Krimpenfort et al.
Lactoferrin Bovine αs1-casein
cattle 1991)
 Improved Disease Resistance
• to treat mastitis, an inflammation of the mammary gland,
typically caused by infectious pathogen(s).
• Mastitis causes decreased milk production.
• Transgenic dairy cows that secrete lysostaphin into their milk
have higher resistance to mastitis due to the protection
provided by lysostaphin, which kills the
bacteria Staphylococcus aureus, in a dose-dependent manner
• Lysostaphin is an antimicrobial peptide that protects the
mammary gland against this major mastitis-causing pathogen
• prion-free (Richt et al. 2007) and suppressed prion livestock
(Efforts for Madcow disease)
Reproductive Performance and Fecundity

• Introduction of a mutated or engineered estrogen

receptor (ESR) gene could increase litter size in a
number of diverse breeds of pigs.
• A single major autosomal gene for fecundity,
the Boroola fecundity (FecB) gene, which allows
for increased ovulation rate, has been identified
in Merino sheep.
• Each copy of the gene has been shown to
increase ovulation rate by approximately 1.5 x.
 Hair and Fiber
• control of the quality, color, yield, and even ease of harvest of hair,
wool, and fiber for fabric and yarn production
• A novel approach to producing spider silk, a useful fiber, has been
accomplished using the milk of transgenic goats. Spiders that
produce orb-webs synthesize as many as seven different types of
silk for making these webs.
• One of the most durable varieties is dragline silk. This material can
be elongated up to 35% and has tensile properties close to those
of the synthetic fiber KevlarTM.
• Its energy-absorbing capabilities exceed those of steel. There are
numerous potential applications of these fibers in medical devices,
sutures, ballistic protection, tire cord, air bags, aircraft, automotive
composites, and clothing.
 Enhancing Growth Rates
• an increase in porcine growth hormone (GH) leads to
enhancement of growth and feed efficiency in pigs
• For fish, there is a need for more efficient and rapid
production, without diminishing the wild stocks, to
provide a protein source for the increasing world
population.
• The production of GH transgenic fish has led to dramatic
(30-40%) increases in growth rates in catfish through the
introduction of salmon GH into these animals.
• Introduction of salmon GH constructs has resulted in a 5-
11 fold increase in weight after 1 year of growth.
 Pitfalls
• possible side effects, e.g., GH transgenic swine had
arthritis, altered skeletal growth, cardiomegaly,
dermatitis, gastric ulcers, and renal disease
• insertional mutations that result in some essential
biological processes being altered
• Mosaicism
• transgene integration on the ‘Y' chromosome,
which results in only males carrying the
transgene.
 Risks
• From the consumer side, the food or
agricultural product produced must be safe,
wholesome, non-allergenic, nutritious, and
economical.
• These are issues being addressed by various
governmental agencies.
• time, cost, welfare, ethics, risks, and benefits
must be weighed