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