Establishment Of The GDF-9Transgenic Mice Model

Purpose:Establish the GDF-9transgenic mice model to further research the function of GDF-9gene.Methods:The pIRES2-GDF9-EGFP vector with Ase I, AflⅡ and Stu Ⅰ three enzymes enzyme loci was contracted. pIRES2-GDF9-EGFP DNA vector were injected into fertilized eggs by means of pronucleus microinjection (PI) to obtain transgenic embryo. Then the transgenic embryos were transplanted into oviduct of the pseudopregnant ICR female mice to obtain transgenic mice. Using PCR and Southern blot to identificate whether the foreign gene was integrated into the FO offsprings, and making the positive transgenic mice mate with the negative ones for reproduction.Results:The result of concentration compare test displayed that the differences of embryo survival rate were not significant between the blank injection group and the other three groups(0.5ng/μL,1ng/μL and2ng/μL), but the four groups had a significantly higher survival rate than4ng/μL injection group (P<0.05). Among DNA injection groups, the differences of cleavage rate and blastocyst rate of the three concentration groups (0.5ng/μL,1ng/μL and2ng/μL) were not significant; but cleavage rate and blastocyst rate of these three groups were significantly lower than the blank injection group (P<0.05); the cleavage rate and blastocyst rate of4ng/μL group were extremly significant lower than blank injection group (P<0.01). The blastocyst positive rate of4ng/μL injection group was44.4%which was significant higher than2ng/μL group (34.1%)(P<0.05) and extremly significant higher than the0.5ng/μL (18.6%) and1ng/μL group (17.1%)(P<0.01). Choosing the concentration of2ng/μL which had low toxicity to embryo and higher EGFP expression rate for pronucleus injection, there were607fertilized eggs were injected and450fertilized eggs were alive and the survival rate was74.1%. All of the16female mice were pregnant after the450surviving embryos transplanted into the oviduct of pseudopregnant ICR female mice and then got95offsprins. The farrowing rate was21.1%. There were three offsprings died in2days after being born. The remaining92offsprings had been tested by PCR, there were4positive transgenic mice. Southern blot detection showed1out of4mice was transgenic positive. There were37offsprings generated by reproduct the F0transgenic mice. The average litter size of the transgenic mice was12.3, which was slightly increased compared to the wild FVB mice.Conclusion:Establishment of GDF-9transgenic mice model revealed that the over expression of GDF-9gene may have certain influence on animal reproductive performance.