Unit 2: Transgenic manipulation Flashcards
Define transgenic
Relating to or denoting an organism that contains genetic material into which DNA from an unrelated organism has been artificially introduced
Outline the steps involved in transgenic mouse production (7)
1) Mate female ‘super ovulated donor’ with male mouse
2) This produces a fertilised egg
3) Pronucleus is microinjected with transgene
4) Transfer embryos into pseudopregnant recipient
5) Identify founder transgenics by DNA analysis from biopsy
6) Mate with wild type
7) See how many of offspring produced are transgenic
What are 3 features of transgenic expression constructs ?
1) Directs tissue specificity of expression
2) Directs time of expression
3) Can be inducible
What is GFP ?
Green fluorescent protein
What is the role of GFP in transgenics ?
- Acts as a reporter gene to track gene expression
- Produces a green florescence to visually monitor when and where a gene is active
What is a promotor ?
A region of DNA that signals the start of the gene and helps to control its expression
Promotors can be inducible, what does this mean ?
It can be turned on or off
What are 2 advantages of transgenics ?
1) Restively fast in vivo assessment of gene function
2) Creates tools for specific gene regulation studies
What are 5 disadvantages of transgenics ?
1) Integration into the genome is random- BIG PROBLEM
2) Integration is often multi copy
3) Site of integration can modify gene expression
4) Large number of animals used to produce TG
5) Commonly performed with a mixed genetic background
What is gene targeting ?
The genetic modification of an endogenous DNA sequence within a cell by homologous recombination with a DNA segment introduced into the cell
In gene targeting, what is positive selection ?
To select for cells that have undergone the desired genetic modification
In gene targeting, what is negative selection ?
To eliminate cells that have not undergone the desired genetic modification
What are the 2 types of gene targeting experiments ?
1) Inactivation of gene (Knock out)- Conditional alleles
2) Introduction of mutations (Knock in)- Promoter analyses
Explain Cre loxP recombination (5)
1) The region of DNA to be targeted (usually an exon) is flanked by loxP sites
2) Cre recombinase gene is introduced
FOR DISRUPTED GENE FUNCTION
3) Cre protein produced in tissue of interest
4) Cre mediated recombination between loxP sites delete intervening DNA
FOR TYPICAL GENE FUNCTION
5) No Cre protein produced in other tissues
What are the 3 requirements for successful gene targeting ?
1) Need isogenic DNA with 4-8kb of homologous sequence
2) The gene must be targeted- lethality
3) Must maintain ES cell totipotency for germ line transmission
