Transgenic animals mice for students

Transgensis: The process of transfer of gene from one organism to another organism.
Transgene: the gene responsible for transfer
Transgenic Mice: can be done by three methods
1)Retroviral Method: by using retroviral vector transgene is inserted into the egg
2) Dna Microinjection: Direct inoculation of transgene into the male pronuclues
3) Embryonic Stem cell: transgene is inserted during embryonic stage of the embryo
There are many applications, limitations .

1. Transgenic animals

2. INTRODUCTION: A transgenic animal is one whose genome has been altered by the transfer of a gene from another species or breed. The foreign gene may be introduced in embryos at the first cell stage. The first transgenic animals were mice created in 1974 by Rudolf Jaenish . The foreign gene is constructed using recombinant DNA methodology. To be incorporated into the DNA of the host To be expressed correctly by the cells of the host

3. STRATEGY: A cloned gene is injected into the nucleus of fertilized egg. The inoculated fertilized eggs are implanted into a receptive female because successful completion of mammalian embryonic development is not possible outside of a female. Some of the offspring derived from the implanted eggs carry the cloned gene in all of their cells. Animals with the cloned gene integrated in their germ line cells are bred to establish new genetic lines.

4. TRANSGENIC MICE: METHODOLOGY Developed in the laboratory mouse. Since the early 1980s, hundreds of different genes have been introduced into various mouse strains. Understanding of gene regulation, tumor development, immunlogical specificity, molecular genetics of development. Examining the feasibility of the industrial production. Act as biomedical models for various human genetic diseases .

5. TRANSGENESIS: DNA can be introduced into mice by, Retroviral vectors that infect the cells of an early-stage embryo receptive female Microinjection into the enlarged sperm nucleus (male pronucleus ) of a fertilized egg. Introduction of genetically engineered embryonic stem cells into an early-stage developing embryo before implantation into a receptive female.

6. 1) RETROVIRAL VECTOR : Advantage : integrating the transgene into the genome of a recipient cell. RNA genome DNA reverse transcriptase can transfer only small pieces ~8Kb Lack essential adjacent sequences for regulating the expression of genes. Drawbacks: Contamination of retroviral vectors Merits: capable of transferring larger fragments, stable for shorter period

7. Lentiviral transfer vector: P – promoter LTR – Long Terminal Repeats at the 5’ and 3‘ end Ψ – packaging RNA into viral particles. PPT – Poly purine tract sequence and WPRE – woodchuck posttranscriptional sequence enhance the transduction of host cells and increase transgene expression in the cells. 5’LTR Ψ PPT P Transgene WPRE 3’LTR Black dot – regulatory element within the 3’end LTR is deleted , prevent the production of vector RNA promoter- not affected by the deletion becoz it expressed by its own promoter

8. transgene with a promoter sequence (p) Inserted into lentiviral vector Transfer vector is introduced into a packaging cell line Produces the viral proteins viral RNA and packaging Deliver transgene into animal cells

9. 2) DNA MICROINJECTION METHOD: Pronucluear method superovulation and mating ( usually 5-10, but here producting 35 eggs) Isolation of one cell stage zygote Microinjection of trangene Oviduct transfer to pseudopregant female

10. 3) EMBRYONIC STEM CELL METHOD: Natural mating Isolation of blastocyst Microinjection of ES cells into blastocyst Uterine transfer to pseudopregnant females

11. DETECTION OF THE TRANSGENE: Detection Methods: Southern Blotting (Tail tip DNA) Provides information on: Number of copies present in each founder line Number of integration sites of the transgene

12. 2) PCR: Can detect low-copy number More rapid than southern blotting Cannot determine copy number or integration sites Best used for screening

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