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

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

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

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)

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

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

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

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

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

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

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.

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

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

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

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

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

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

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

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