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.
1) To be incorporated into the DNA of the host
2) To be expressed correctly by the cells of the host

3. STRATEGY:
1) A cloned gene is injected into the nucleus of fertilized
egg.
2) The inoculated fertilized eggs are implanted into a
receptive female because successful completion of
mammalian embryonic development is not possible
outside of a female.
3) Some of the offspring derived from the implanted eggs
carry the cloned gene in all of their cells.
4) 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,
1. Retroviral vectors that infect the cells of an
early-stage embryo receptive female
2. Microinjection into the enlarged sperm
nucleus (male pronucleus) of a fertilized egg.
3. 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

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

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

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