Study On Sex Identification Of Mouse Preimplantational Embryos And Embryonic Fibroblasts

Sex identification of early stage in mammal embryos is one of the methods for sex control, by which to build efficient stock commercially, to take advantage of maternal productivity and paternal physical superiority, as well as genetic harm limitation and sex-linked diseases prevention. The research is also the key point to molecular biology, embryonic cytology and developmental genetics. MEFs are a practical method in ESCs culturing, somatic cell cloning and nuclear transferring for it is enormous and accessible in vitro with rapid growth and n long-term generation. On this research, mice preimplantational embryos and MEFs were sex-identified by double PCR for ESC culturing, somatic cell cloning and nuclear transferring. Specifically:(1) Primer design and its reasonableness test:designed male-only SRY gene primers:SRY250U:5’-CTTTTTCCAGGAGGCACAGA-3’and SRY250L: 5’-GACAGGCTGCCAATAAAAGC-3’; general ZFX gene primers with zinc finger structure:ZFX399U:5’-AAGAGAGTCCATTCAAGTGTGA-3’and ZXF399L: 5’-GCTACCTTTGTTGCCGAAAT-3’. Use centrifugal pillar type animal genomic DNA extracted kit extraction mouse liver tissue DNA genome as a template. In the appropriate reaction system and conditions, amplified two primers respectively in vitro; checked the results with 2%agarose gel electrophoresis. The results shows that DNA from male mice can be amplified the 250bp judgmental strip with SRY gene primer while results from females are negative; with ZFX gene primer, both male and female mice’s DNA can be amplified to 399bp products. Amplified DNA from liver cells of specific sex with both SRY gene and ZFX gene primers, the results shows that male mice are both positive while female mice have 250bp products only.(2) 2.5d embryos were gathered from mice injected PMSG and hCG, erased zona pellucid with 0.5%pronase at37℃,30s.Separated 4 of 8 cells from embryo, added 8μL ddH2O, Constant temperature 5 min after boiling 20℃ice bath 5 min,14000 rpm centrifugal 2 min, take clear liquid used for the amplification, amplification system for: ddH2O 7μl; 10 by Buffer 3μl; Mg2+2μl;10 by Taq Buffer 2.5μl; dNTP 3μl; Primer P1 1μl; Primer P21μl; DNA sample 5μl; Taq enzyme 1.5μl; Reaction conditions for:the degeneration 94℃,5 min; Degeneration 94℃,30 s; Annealing 60℃,35 s; Stretching 72℃,40 s, a total of 30 circulation; 72℃,10 min,4℃发preservation. After the expansion take 5μl products in 1.5% agarose electrophoresis on examination results.40 embryos were identified,20 of which are male with 250bp and 399bp results, 12 of them are female with 399bp results. Another 8 embryos is fail to being identified for blur strips. The rate of identification is 80.00%.(3) 13.5d embryos were gathered from mice injected PMSG and hCG to get MEFs. Will the MEFs cellular density of adjustment to 1 x 106 a/ml, in 37℃,5%CO2, cultivate, every 24 h refreshed. Resulted that F3 MEFs is the best. With 80-90%rate, took the F3 generation MEFs by centrifugal column type cells genomic DNA extracted DNA extracted kit total; Use the SRY works independently design primer and ZFX genes to MEFs DNA primers to expand in vitro, amplification system for:ddH2O 7μl; 10 by Buffer 3μl; Mg2+2μl;10 by Taq Buffer 2.5μl; DNTP 3μl; Primer P1 1μl; Primer P21μl; DNA sample 5μl; Taq enzyme 1.5μl; Reaction conditions for:the degeneration 94℃,5 min; Degeneration 94℃,30 s; Annealing 60℃,35 s; Stretching 72℃,40 s, a total of 30 circulation; 72℃,10 min,4℃preservation. After the expansion take 5μl products in 1.5%agarose electrophoresis on examination results. 30 fetal mice were indentified,13 of which are male with both 250bp and 399bp results; 7 of them are female with 250bp only.10 of MEFs are not able to be identified for missing strips. The rate is 66.67%.In research, male SRY and ZXF gene primers are designed and rationalized to identify sex of preimplatational embryos and MEFs with double PCR successfully, achieving rate by 80.00%and 66.67%respectively. The results lead to that 4 of 8 cells from embryos is optimized, and MEFs isolated from 13.5d fetal with better growth are advisable to be practiced in ESCs culturing, somatic cells transferring as well as embryo transferring.