Studies On Construction Of Somatic Cloned Embryos And Expression Of Ercc6l Gene In Sika Deer

Many remarkable progress has been made in mammal somatic nuclear transfertechnology (SCNT). After the first cloned "Dolly" was born in1997,19differentkinds of mammals has been obtained successfully cloned offspring through somaticnuclear transfer techniques, throughout the world set off a "clone" craze. Sika deer is avery rare animals, it has a close relationship with human life. It can provide moreexpensive medical care for human medicines, can also provide humans with food andclothing. We are still seeing reports of sika deer in SCNT. This thesis is mainly onSCNT and related issues in sika deer, which is included oocyte maturation in vitroculture conditions optimization, parthenogenetic activated oocytes TSA treatmenteffects on the development of cloned embryos, the construction of cloned embryos bydemecolcine-assisted and colchicine-assisted enucleation, development-related Ercc6lgene cloning and molecular testing. This thesis is to improve the efficiency ofpreimplantation embryos by SCNT in sika deer. To establish procedures for theproduction of cloed sika deer has laid a solid foundation.This experiment of the sika deer's oocytes matured in vitro conditions areoptimized:(1) comparison of sika deer in mating season oocytes in7different media(media0-6) in vitro maturation rate of20h and24h;(2) in vitro maturation of oocytesin culture medium6in estrous season and anestrous season;(3) optimization of invitro maturation of oocytes by hormones added in the anestrous season. Looking forsika deer in estrous season and anestrous seasons of the best conditions of in vitromaturation of oocytes, compared the mature rates of change under the condition ofdifferent media. Results indicate that mature rates of the oocytes in the media6(M199+10μg/ml FSH+1μg/ml LH+0.2mM Cys+50ng/ml EGF) is up to75.4%, are significantly higher than in medium0,1,2, but are not significantly higher than inmedium3,4,5in the estrous season. β-estradiol does not increase the rate of oocytematuration. However, double concentration of homocysteine can significantlyincrease the rate of oocytes maturation in vitro. After extended4h in vitro culture,mature rates of the oocytes in24h are slightly higher than those in20h, but thedifference is not significant. Maturation rate of oocytes in estrous season issignificantly higher than this anestrous season. Double FSH, LH, cysteine and EGFcan significantly improve the rate of oocyte maturation of sika deer in anestrousseason.Study on Parthenogenetic activated oocytes of sika deer:(1)220V/mm-260v/mmcurrent stimulation on the development of parthenogenetic embryos;(2) Comparisonchemical activated effects of ethanol+6-DMAP and Ionomycin+6-DMAP on theparthenogenetic embryo development; compared Ionomycin+6-DMAP obtainedparthenogenetic blastocysts and in vivo fertilization blastocysts in quality. Resultsindicate that the220V-260V stimulated by different voltages,230V electricalstimulation can be a little high rate in cleavage, morula and blastocyst. Chemicalactivation can significantly enhance the development of parthenogenetic embryos ofsika deer. Chemical activation of Ionomycin+6-DAMP is significantly higher thanethanol+6-DAMP in the rate of cleavage (72.7%vs.61.1%), morula (43.9%vs.29.7%) and blastocyst (32.4%vs.17.8%). There were no significant differences forIonomycin+6-DMAP obtained parthenogenetic blastocysts and in vivo fertilizationblastocysts on the ICM, TE, total number of cellsTSA treatment effects on the development of cloned embryos:(1) Established earfibroblasts and fetal fibroblast cell line and transgenic pEGFP-N1fetal fibroblast celllines;(2) Effects of different concentrations of TSA on the donor cells;(3) differentconcentrations of TSA treated immune fluorescent staining of H4K5of pEGFP-N1fetal fibroblasts;(4) TSA treatment of different concentration on early development ofcloned embryos;(5)50nM TSA treated three kinds of donor cells on earlydevelopment of cloned embryos. Results show that when a concentration greater than50nM TSA, cells have significantly changed cells are no longer as fusiform and become flat. When concentration of TSA is100nM, significantly inhibited cellproliferation, cell started a small number of deaths. When the TSA is greater than200nM, and a lot of cell start death. H4K5protein signal increases as the TSAconcentration increases in transgenic pEGFP-N1fetal fibroblast cell lines. The rate ofblastocyst is available16%in50nM TSA treated transgenic pEGFP-N1fetalfibroblast cell lines as the donor somatic cell, significantly higher than other groups.Reprogramming capability of fetal fibroblast cells as donor cells is larger than this offibroblasts as donor cells, which is more suitable for somatic cell nuclear transfer.The construction of cloned embryos by demecolcine-assisted andcolchicine-assisted enucleation:(1) effect of colchicine and demecolcine in differentconcentrations treated oocytes to form cytoplasmic protrusions;(2) effects ofcolchicine and demecolcine formed cytoplasmic protrusions in oocytes age;(3)persistence of the demecolcine-induced cytoplasmic protrusions;(4) effects of oocytecumulus expansion on demecolcine-induced cytoplasmic protrusions;(5) changes inthe position of the FPB in oocytes treated with demecolcine;(6) development ofcloned embryos in SOFaa and DSOF;(7) development of cloned embryos afterdemecolcine-assisted, colchicine-assisted and blind enucleation. Results indicate thatwhen oocytes were incubated in TCM199containing0.8μg/mL demecolcine for1h,95.93%of the oocytes developed protrusions. when oocytes were incubated inTCM199containing10μg/mL colchicine for4h,90.1%of the oocytes developedprotrusions. Demecolcine-assisted enucleation is reversible process, good quality ofoocytes formed cytoplasmic protrusions are higher. However, colchicine-assistedenucleation is not reversible process, old oocytes formed cytoplasmic protrusions arehigher. Demecolcine-assisted enucleation and colchicine-assisted enucleation is morecomparative advantages than blind enucleation on the development of preimplantationembryos.Development-related Ercc6l gene cloning and molecular testing:(1) cDNAcloning and sequence analysis of sika deer ercc6l;(2)5′and3′Rapid amplification ofcDNA ends (RACE);(3) sequence analysis, multiple sequences alignment, andphylogenetic analysis;(4) quantitative real-time PCR (qRT-PCR) analysis of Ercc6l expression in different developmental stages of cloned embryos;(5) qRT-PCRanalysis of Ercc6l expression in six organs at six different developmental stages.Results indicate that Ercc6l gene mRNA (registration number: HQ529500) sequenceis4197bp, which include51nucleotides5' UTR,414-nucleotide3' UTR, and polyAtail12nucleotides, which included include3,732nucleotide composition of openreading frame coding1243amino acids in sika deer. Ercc6l expression ofparthenogenetic embryos and cloned embryos differences are not significant indifferent developmental stages. The highest Ercc6l expression levels in every organsample were seen at three months old. However, the lowest Ercc6l expression levelswere seen at three years old in every organ.