1. Definition: Transgenic Animal Animal in which a segment of DNA has been physically inserted into the genome. The genome of all cells of the organism contains the DNA segment. The DNA segment is present in the germ line so it can be transmitted to progeny as a Mendelian trait.

2. Retroviral Mediated Gene Transfer MoMuLV Producing Cells Mouse embryos Insertion of viral DNA containing transgene into embryos Transgenic Mouse

3. X 1-cell pronuclear stage mouse embryo. PMS + HCG to superovulate  Pregnant Foster Mother Oviduct Transfer + + 2-cell stage embryo Holding Pipette Injection Pipette

4. Events During Transgenesis DAY Fertilization 12 AM12 AM12 AM12 AM12 AM12 AM12 AM Reimplantation Mating Microinjection Cell Division Gestation Birth Embryo Recovery E1E2E21 12345 hCGPMS

5. Embryonic Stem Cells Pluripotent stem cells isolated form the inner cell mass of a blastocyst stage mouse embryo. Cells retain ability to differentiate into all organs and cells. Cells can be propagated under conditions in vitro that allow them to retain their totipotency- prevent differentiation in vitro. Can be induced to differentiate in vitro into a number of cell types. ES cells can mimic early mammalian development in vitro. Can be manipulated in vitro to alter the genome through homologous recombination.

6. ES cell CultureTargeted ES Cells Pure Population of Targeted ES Cells Targeted ES Cells Injected into Blastocyst  Pregnant Foster Mother X Chimera Blastocyst derived from mouse with white coat. Breed to WT white mouse Germline transmission of ES cell-derived genome. Uterine Transfer

7. A B C D E F G H I B C DE F G X A B C D E F G B C D E F G H I Gene Knockout by Homologous Recombination Using an “O” or “Insertion” Type Construct neo

8. A B C D E F G H I B CF G XX A B C F G H I Gene Knockout by Homologous Recombination Using an “  ” or “Replacement” Type Construct neo TK neo