Use of Transgenic Mice in Reproductive Biology

Generation of transgenic mice by oocyte pro-nuclear microinjection + gene targeting in ESC easy in mice

Gene Driven Studies--reverse genetic studies; interested in gene; what happens to phenotype

Phenotype driven studies--forward genetic studies; interested in phenotype; what gene caused it

Random integration

don't mind where transgene inserts

find where gene expressed

transgene with promoter for gene of interest upstream to reporter gene

Constitutively expressed or cell-specific promoter

Promoters--intronic or 3 end regulatory sequences; combined with reporter; cutting with restriction enzymes; paste with DNA ligase into bacterial plasmid; amplified in E.coli

Reporter genes e.g. GFP; B-galactosidase

Nestin-GFP in testicular leading cells to identify precursors

B-galactosidase; stain with Xgal; Pygo2-LacZ expression in whole mount and thin tissue segments

Inject oocyte with transgene-->pseudopregnant mice; ear notch to genotype via PCR

Gene Targeting

Knock-in

Knock out

Target vector construction: vector contains homologous DNA to target gene + inserted DNA to change target gene + marker gene + cell death gene

Neomycin--marker gene; between homologous sequences

HSV-tk--cell death gene; outside homologous sequence

Produce transgenic mice; cut transgender from bacterial plasmid then lipid-based transfection
or electroporation

ESC selection

neomycin resistance or HSV-tk cell suicide

Transgene into blastocyst; mosaic; pseudopregnant; chimeric offspring x normal mice; gene targeted mice

Remove chimera step: tetraploid embryos; fusion of 2x2-cell embryos WT; inject transgene ESCs; tetraploid becomes trophoblasts; ESCs become embryo

E.g. CatSperm proteins + androgen receptors in testes

Cre-LoxP Contional Knockout

Disadvantages of Gene targeting: embryonic lethality, incompatible with life, developmental problems due to genetic deletion

recombinase viral system

Androgen receptor in testicular cells;

2 mouse cell lines crossed

floxed; contains loxP target site; specific target insertion; before and after target gene

Cre-enzyme line; random insertion; cuts at loxP; enzyme expression enhanced by promoter

Crossing cell lines--conditional KO offspring

Sertoli Cell Specific ARKO--AMH-Cre from E14.5--infertility; blocked spermatogenesis at meiosis

PTMC-specific ARKO--MHC-Cre in PTM + mesenchymal cells--infertility with reduction of sperm at all stages

Leydig Cell Specific ARKO--Ap2-Cre; Leydig cells + precursors; not infertile; LCs don't develop properly + undergo apoptosis in adulthood

CRISPR/Cas9 system

bacterial system; GM organisms that conventional methods don't work on

Cas9 RNA guided DNA endonuclease

Basic premise; induce ds break at somewhere on genome and allow cell repair mechanism to fix

non-homologous end joining: INDELS to cause mutation; no template DNA

Homology-directed repair: donor template ; needs guide RNA, mRNA for Cas9 and donor template