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Transgenesis
 Transgenesis
• In transgenesis, foreign desired gene is added to animal
genome for desirable character
• Genetically modified organisms (GMO): organisms (plants,
animals, bacteria and viruses) who’s genome have been
deliberately altered through the transfer or deletion of
genes
• GMO also transfer character to offspring
• Transgene- gene transferred naturally or by genetic
engineering from one to other organism
History
• 1983- transgene was added to tobacco by Richard B.
Flavid and Marry dell.
• 2000- Vit. A enriched rice developed
• 2001- silk producing goat was produced
• vaccine containing banana
• Blue roses
• Oncogenic mouse or Harvard mouse (carry cancer causing
gene)
 Advantages
• Research
• Heritability of transgene
• Genetic diseases by gene therapy
• Disease resistance by antibody production
• blood replacement, Alzheimer, cardiovascular, cancer,
diabetes mellitus, angiogenesis
 Advantages
• Xenotransplantation- organ transplantation from one species to other
• Safety testing
• Pharmacy/Pharmaceutical production/toxicity check in animal model
• Biological and medical research
• Agriculture- food scarcity, plant crossing with cold water fish to
ensure year round availabilty of seasonal crops.
• more specific, rapid, flexible (entry of genes not already present), less
time required
• Mammals development genetics, gene expression study, genetically
engineered hormones etc
• Detect, diagnose and treat diseases
•
 Advantages in Livestock
• Better FCR
• Desired carcass composition
• Milk composition modification
• Reproductive performance and prolificacy
• Environment friendly transgenic animals
• Modifications in wool, fiber and hair quality, color, yield,
season of collection and even ease of harvest
• Transgenic animals in immunology, oncology, genetic
engineering, gene thrapy and as model in himan diseases
 Disadvantage
• Low efficiency
• Precise gene expression
• Time and cost
• New viruses/pathogens entry
 Disadvantage
• Food safety
• Biodiversity
• Ethical issues
• Human interaction with animals
• Biopiracy
• Intellectual properties and dominance of developed nations
on underdeveloped
• Labelling issues i.e. in some countries as USA is not
mandatory so issue of identification of GMO and non GMO
will arise.
 Gene Preparation
• Gene identification
• Gene isolation/Editing
• Cloning of desired gene
 Gene Identification
• Gene identification by 1. gene chips/ microarrays 2. DNA
sequencing by PCR
 Gene Editing Techniques
• Recombinant DNA technology
• Zinc Finger Nucleases (ZFNs)- protein (from xenopus
oocyte) based DNA target system
• Transcription activator-like effector nucleases (TALENs)-
Bacterial protein based DNA target system- more efficient
as ZFNs
• CRISPR/cas9 (clustered regularly interspaced short
palindromic repeat) Bacterial protein based DNA target
system in defense system
 Recombinant DNA technology
• Cutting and splice DNA fragments together in laboratory
resulting in recombinant DNA that cause specific gene
expression
• Steps- 1. gene isolation by restriction enzymes 2. cloning or
insert of desired gene in plasmid (chimera) 3. chimera
introduction in host cell (transformed cell) through heat
shock (42 ̊ C for 2-5 minutes) and electroporation and
vectors
 Zinc Finger Nucleases (ZFNs)
• ZFNs (discovered in 1985) are assembled by fusing to a site
-specific DNA-binding domain loaded on the zinc finger.
• The zinc-finger comprises an array of Cys2His2 zinc fingers
(consists of approximately 30 amino acids), derived by zinc
-finger attachment with homologous DNA sequences. The
FokI type II restriction endonuclease cleaves the DNA,
creating the desired alterations in genome by endogenous
NHEJ (nonhomologous end joint ) or HDR (homologous
directed repair) repair systems
• The target sequence recognition and specificity of ZFNs
are determined by three major factors: (a) the amino acid
sequence of each finger, (b) the number of fingers, and (c)
the interaction of the nuclease domain.
 Transcription activator-like effector
nucleases (TALENs)
• Working is similar to ZFNs
• Talen's source is different (33-35 amino acids)
• limit is 28-40 TALEN bp
• Repeat variable diresidues-RVDs


Clustered Regularly Interspaced Short
Palindromic Repeat (CRISPR/CAS9)

• CRISPR/Cas9 system comprises two components, a single-
stranded guide RNA (sgRNA) and a Cas9 endonuclease.
The sgRNA complement the target DNA site, followed by a
short DNA sequence on upper side (termed protospacer
adjacent motif-PAM) essential for the compatibility with the
Cas9 protein that cleaves the DNA to generate a DSB. DNA
-DSB repair mechanism through NHEJ or HDR resulting
targeted genomic modifications.
Advantages of CRISPR/CAS9
• ZFN or TALEN demand reengineering of the nuclease
enzyme each time however, the nuclease protein Cas9 is
identical in all cases and need not each time reengineering.
• In contrast to CRISPR/Cas9, ZFNs and TALENs demand
more labor and are expensive.
• CRISPR/Cas9 has the potential of simultaneous multiple loci
editing, making this technology easier and more efficient.
 Procedure of Foreign Gene Insertion
• Cloned gene transfer in host- chimera introduction in host
cell (transformed cell) through heat shock (42 ̊C for 2-5
minutes) and electroporation→ desired gene entry in host
c e l l t h r o u g h v e c t o r s, v ir u se s, g e n e g u n an d DN A
microinjection
 Gene Insertion
• Heat shock and electroporation
• Retroviruses (70s)-size of foreign DNA, viral replication,
virus interference with gene expression etc.
• Gene gun
• Microinjection
• Liposomes
 Gene Incorporation Vectors
• A small piece of DNA containing foreign DNA with ability to
replicate itself for transferring or propagating in an organism
is called transgenic/cloning vector. Vectors increase
probability of gene expression
 Gene Incorporation Vectors
• Plasmids vectors- small, naturally occurring, circular and
extrachromosomal pieces of DNA isolated from bacteria (10
-700 copies) and are restricted to accept DNA up to 5000
base pairs, most common used plasmid is pUC18
• Bacteriophage- one third of their genome is free and can
c ar r y f or e ign ge n e DN A u p t o 1 0 - 1 5 kilobase s i. e .
bacteriophage lambda
 Gene Incorporation Vectors
• Cosmides- have properties both of plasmids and
bacteriophages and can carry DNA segments up to 50
kilobases
• Yeast artificial chromosomes- prepared to carry up to 1
million base pairs of DNA strands
 Gene Incorporation Vectors
• Liposome- a small membrane bound vesicle that can
enclose vector and transfer foreign gene by binding to cell
or nuclear membrane of host cell
• Transposons
 Transposons
• Transposons are short genomic DNA regions (1-3kb) that
auto replicate and randomly integrated as multiple copies
within same genome. This integration is carried out by
integrase enzyme
• Foreign DNA is added in transposon and recombinant
transposon is then microinjected in single cell embryo
• This is used in insects, silk worm, chicken, fish and
mammals
 Transgenesis Methods/Sources
• Gonads
• Sperm
• Transgenesis by transgene transfer to zygote/fertilized egg
and embryo via DNA microinjection, retroviruses (carry
small genome, can cause new pathogens and unable to
replicate in early embryo are its limitations) and stem cells -
desired gene are entered in stem cell and then in host cell
(need not live animal for transgene verification)
• SCNT/Cloning
DNA Microinjection- Method & Limitations

• Method- gene identification→ isolation → cloning → gene

insertion invitro in male pronuclei before fusion → embryo
transfer in recipient → transgene verification in offspring
• Limitations- low success rate (1-4% offspring livebirth
containing transgene), laborious and costly, in lower
vertebrates and invertebrates and non mammals pronuclei
is not visible and transgene is added in cytoplasm and
require large DNA quantity
 Transgenesis Success Verification
• Incorporation of antibiotic resistant gene/enzymes-
aminoglycosides as neomycin and kanamycin
• Western and southern blot
• PCR
• ELISA
 Transgenesis Success Verification
• Green fluorescent protein (GFP) – ideal transgenesis
marker
• β galactosidase - transgenesis marker
• firefly luciferase- transgenesis marker
• Placental alkaline phosphatase - transgenesis marker
• Questions Please if Any?

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