Transgenic animal production methods.

1. TRANSGENIC Technology
Purnima Kartha. N

2. Transgenesis
• Transgenesis consists of introducing an exogenous DNA
sequence into the genome of a pluricellular organism,
which then becomes present in most cells and is
transmitted to progeny.
• Transgenesis is different from gene therapy. In the latter
case, the germ cells do not harbour the foreign DNA.

3. • The DNA fragments used to generate transgenic organisms
are genes containing a sequence preceded by a promoter
driving its expression to an RNA or a protein.
• The gene transcript may be an RNA not translated into a
protein : Antisense RNA, ribozymes etc.
Transgenesis

4. Aim of transgenesis
To add foreign genetic information to a genome
To suppress an endogenous gene
To replace a functional gene by another functional gene
The foreign gene may be a mutant of the endogenous gene or a
completely different gene.

5. Applications of Transgenic Technology
Genetic bases
of human and
animal
disease-
Design &
Testing of
therapy.
Disease
resistance in
humans and
animals.
Gene therapy
Drug and
product
testing and
screening.
Novel product
development
through
“molecular
pharming”.
Production
agriculture.

6. Embryonic stem cell method

7. Embryonic stem cell method
o In the ESC method, cells at Blastocyst stage are proliferated in a cell culture.
o Foreign DNA is inserted into the ESCs by electroporation or microinjection
method and the desired segment is identified with the help of PCR and marker
genes.
o The gene construct of interest is fused to blastula cells and the modified blastula
is implanted into the host mother.
o The offspring obtained from this are mated with normal animals to obtain
transgenic species.
o The advantage of this technique is that the DNA of interest can be inserted to the
exact locus.
o First offspring produced from such animals are chimeras.
o True transgenic animals are obtained in the second generation.

10. Pronuclear microinjection
• Pronuclear microinjection involves the direct transfer of DNA into the male
pronucleus of the fertilized mouse egg.
• Just after fertilization, the small egg nucleus (female pronucleus) and the large
sperm nucleus (male pronucleus) are discrete.
• Since the male pronucleus is larger, this is usually chosen as the target for
injection.
• About 2 pl of DNA solution is transferred into the nucleus through a fine needle,
while the egg is held in position with a suction pipette.

11. Pronuclear microinjection

12. • The injected embryos are cultured in vitro to the morula stage and then transferred
to pseudopregnant foster mother.
• The procedure requires specialized microinjection equipment.
• The resulting animal may be transgenic or may be mosaic for transgene insertion.
• The technique is reliable, although the efficiency varies, so that 5–40% of mice
developing from manipulated eggs contain the transgene.

13. • Once the transgene is transmitted through the germline, it tends to be stably
inherited over many generations.
• The exogenous DNA tends to form head-to-tail arrays prior to integration, and the
copy number varies from a few copies to hundreds.
• The site of integration appears random and may depend on the occurrence of
natural chromosome breaks.

14. Retroviral Vector Method
• This technique has generally been used to produce transgenic mice where DNA
fragments of small size (8 kb) could effectively be transferred.
• Drawback:
▫ Retroviral contamination, which can interfere in the signal that determines
expression of the inserted gene.
▫ Risk of losing regulatory sequences.
• It is therefore not a commonly used method

15. • Recombinant retroviruses provide a natural
mechanism for stably introducing DNA into
the genome of animal cells.
• Retroviruses are able to infect early embryos
(as well as ES cells), so recombinant
retroviral vectors can be used for germline
transformation.
• An advantage over the microinjection
technique is that only a single copy of the
retroviral provirus is integrated, and the
genomic DNA surrounding the transgenic
locus generally remains intact.