1. Dr. Peter John M.Phil, PhD Atta-ur-Rahman School of Applied Biosciences (ASAB) National University of Sciences & Technology (NUST)

2. Genetic Manipulation of Animals
To insert a foreign DNA artificially into cells of an animal, produced a transgenic animal
Advantages:
To study gene function: The ability to insert genes into whole animals or to selectively delete or alter single predominated genes in an animal provides enormous power in studying gene function
Animal Model of disease: By generating animal model we can study different diseases/disease genes

3. Germ line DNA
To create genetically modified animals it is necessary to modify DNA of germ line cells so that modified DNA is heritable
Certain cells that have capacity to differentiate into the different cells are considered to be the target for introducing foreign DNA

4. Totipotent Cells
The fertilized Oocyte is one such cell, being totipotent.
Other target cells are cells of very early stage embryos, including embryonic stem cells (ESC)

5. Transgenic Animal
Transgenic: Transfer of foreign DNA into totipotent/pluripotent E Cells (fertilized oocyte, very early embryo cell, cultured emryonic cells)
Transgenic Animal: foreign DNA intergrate into chromosomes of a fertilized oocyte, all nucleated cells contain DNA, it is fully transgenic

7. Mosaic
Mosaic:
If foreign DNA insertion occur at post zygotic stage, some cells containing tans gene other lacking it
PCR test can be used to screen for the presence of trans gene

8. Gene Targeting
Sometimes trans genes affect the endogenous gene expression (insertional mutation)
Gene Targeting: A method/process by which mutation introduced into preselected endogenous gene.
It can be achieved in somatic cell (any cell of a living organism other than the reproductive cells)
In ES cells this is powerful, in which all nucleated cell contain mut at the desired locus

9. Strategies for Transgenic Animals
Transfer of cloned DNA into fertilized Oocytes & Cells from very early stage embryo
Pronuclear microinjection
Transfer into Pre/Post implantation embryo

10. Pronuclear microinjection
Superovulated female, mated to fertile male & sacrificed next day
DNA is microinjected into male pronuclear of individual oocytes.
Surviving oocytes are reimplanted into the oviduct of foster female
Microinjected DNA randomly integrate into chromosomal DNA & can be checked by PCR

12. Transfer into Pre/Post Implantation embryo
Early embryo cells (totipotent) can provide a route for foreign DNA to enter into germ line
Foreign DNA can be integrated by retroviruses
Retroviruses can integrates at random

13. Transfer into Pre/Post Implantation embryo
The virus particle contains the RNA genome and viral reverse transcriptase within an outer lipoprotein envelope and inner protein capsid.
A double-stranded DNA copy of the viral genome integrates into the host DNA.
Here it directs synthesis of viral RNA and proteins, which self-assemble and bud off from the cell membrane. The host cell is not killed.

15. Cultured Embryonic cells
Transfer of foreign DNA into cultured embryonic cell is most appropriate way
blastocyte is structure formed in early embryogenesis It possesses an inner cell mass, or embryoblast which subsequently forms the embryo
ES cells are derived from of the blastocyte & cultured in in vitro
These cultures cells injected back into host blastocyte & reimplanted in a pseudopregnant mouse

16. Cultured Embryonic cells
Chimera: The developing embryo is chimera which contain two populations of cells derived from different zygotes (from blastocyte & implanted ES cells)
Use of genetically modified ES cells result in partially transgenic mouse, fully transgenic mice can derive by mating the offspring

17. Cultured Embryonic cells Advantages
ES cells can be grown readily in culture
The desired genetic modification can be verified in tissue culture
The presence of desired gene can be verified by PCR based assay
Powerful approach for understanding gene function & creating animal model of disease

19. Application of TG Animals
Provide a sensitive method of detecting gene expression and regulation of gene expression
For the study of gene function by targeted gene inactivation
Valuable information can be gained by over expression of a gene

20. Application of TG Animals
Transgene can be designed consisting of a tissue specific promoter coupled to a sequence encoding a toxin, for cell lineage ablation
Certain gene that produce gain of function mutation can be investigated by this
For animal model of human disease

21. Creating animal model of disease Human/Mouse difference
Biochemical Pathways
Development Pathways
Absolute Time
Genetic Back Ground

22. Animal Cloning Clone: Genetically identical molecules
Identical twins are clones
Nuclear transfer technology is used in this case
In this case clone don’t share mitochondrial DNA like identical twins

24. Thanks