PCR方法鉴别早期胚胎性别及相关技术的研究
摘要
本论文研究了小鼠和牛早期胚胎的徒手切割取样方法、切割取样胚胎冷冻一解冻方法和PCR方法鉴别牛早期胚胎性别技术，并应用牛Y—染色体特异引物PCR扩增牛、牦牛、山羊、绵羊、小鼠、猪等6个动物基因组，克隆测定扩增产物的序列。试验结果表明：
1．体视显微镜下，应用玻璃切割针徒手切割取样小鼠和牛胚胎；切割取样后胚胎体外培养48小时的发育率分别为76.7％(46／60)和80％(16／20)，牛新鲜胚胎和冷冻—解冻胚胎切割取样后移植妊娠率分别为47.1％(8／19)和42.9％(6／17)。
2．分别以10％甘油、10％乙二醇作为冷冻液以及在以上两种冷冻液中分别添加10％葡聚糖+0.1M蔗糖作为冷冻液常规冷冻方法冷冻切割后的小鼠胚胎，冷冻—解冻后体外培养72小时总发育率分别为56.9％(259／455)，81.8％(317／)，84.8％(540／738)和59.5％(308／518)。取样后胚胎体外短暂培养(0—4小时)并不能提高切割取样后小鼠胚胎冷冻一解冻体外培养发育率。
3．切割取样小鼠胚胎快速冷冻体外培养发育率为73.3％(33／45)，切割取样牛胚胎在25％甘油+0.25M蔗糖+20％血清的PBS为冷冻液快速冷冻后移植妊娠率为57.1％(4／7)。
4．依据牛Y染色体特异序列设计合成5对Y染色体特异引物，依据牛骨胳肌α肌动蛋白前体基冈和微卫星DNA序列设计合成4对牛DNA特异内标引物。应用合成的引物单重PCR扩增牛血样DNA，筛选到4对牛Y染色体特异引物和一对牛DNA特异内标引物。应用所筛选的4对牛Y—染色体特异引物和内标引物分别PCR扩增牛基因组DNA、成纤维细胞和胚胎，筛选出2个可用于牛胚胎性别鉴别的多重PCR引物组合：B34／A12、B78／A12。建立了两温度循环PCR方法(94℃变性15s和55℃退火15S，以94℃变性1s和55℃退火1s)并鉴别牛胚胎性别，将PCR扩增时间减少到50分钟左右。
5．设计合成的5对牛Y—染色体特异引物都可以扩增牛和牦牛Y—染色体，引物B90在绵羊中也有扩增产物，其他动物均未获得扩增产物。扩增产物序列测定结果表明，2对牛Y—染色体特异引物(引物B90、B12)所扩增牦牛的产物序列为已知序列，2对牛Y—染色体特异引物(引物B78、B34)所扩增牦牛的产物序列为新序列。
The splitting and embryo cells sampling techniques of mouse and bovine pre-implantation embryos, the cryo-preservation of split-sampled mouse and bovine embryos , and the sexing of pre-implantation bovine embryos by PCR were studied systematicly. Genomic DNA of six farm animal species such as cattle, yak, goat, sheep, mouse and pig were amplificated by PCR with bovine Y-chromosome specific primers in this study, and the conservative sequences in different species were cloned and sequenced. The results were as follows:
1. Simple technique of mouse and cattle embryos splitting and embryo cells(5-10cells) sampling under stereo-microscope with glass needle or metal razor blade. was established. Development rates of split-sampled mouse and bovine embryos culture in vitro for 48 hours were 76.7%(46/60) and 80%(16/20), respectively. The pregnant rate of recipients of split -sampled bovine fresh and frozen-thawed embryos after transfer were 47.1% and 42.9%, respectively.
2. The total development rates of split-sampled mouse embryos in vitro after frozen-thawed by traditional freezing method in 10% Glycerol, 10% ethylene glycol, 10% glycerol + 10% dextran + 0.1M sucrose and 10% ethylene glycol+ 10% dextran + 0.1M sucrose freezing solution were 56.9%(259/455), 81.8% (317/), 84.8% (540/738) and 59.5% (308/518) , respectively. Short time (0-4hours ) in vitro culture of splitted angd sampled mouse embryos before freezing did not significantly effect on their development rate after frozen-thawed..
3. Quick-freezing technique of split-sampled mouse and bovine embryos was established. The total development rate of quick-freezing split-sampled mouse embryos culture 48 hours in vitro was 73.3% (33/45) ,. The rate of pregnant recipients of frozen-thawed split-sampled bovine embryos, frozen in 25% Glycerol + 0.25M sucrose +20%serum PBS, was 57.1%
(4/7) .
4. Five pairs of bovine male-specific PCR primers were designed based on the sequences of bovine Y-specific repeat sequence, testis-specific protein gene from Y-chromosome (TSPY) and sex determining region Y-protein gene (SRY) from GenBank. Meanwhile, based on skeletal alpha action precursor gene and Bovine 1.715 satellite DNA, 4 pairs of bovine-specific PCR primers were designed as internal control primers. After PCR
1．体视显微镜下，应用玻璃切割针徒手切割取样小鼠和牛胚胎；切割取样后胚胎体外培养48小时的发育率分别为76.7％(46／60)和80％(16／20)，牛新鲜胚胎和冷冻—解冻胚胎切割取样后移植妊娠率分别为47.1％(8／19)和42.9％(6／17)。
2．分别以10％甘油、10％乙二醇作为冷冻液以及在以上两种冷冻液中分别添加10％葡聚糖+0.1M蔗糖作为冷冻液常规冷冻方法冷冻切割后的小鼠胚胎，冷冻—解冻后体外培养72小时总发育率分别为56.9％(259／455)，81.8％(317／)，84.8％(540／738)和59.5％(308／518)。取样后胚胎体外短暂培养(0—4小时)并不能提高切割取样后小鼠胚胎冷冻一解冻体外培养发育率。
3．切割取样小鼠胚胎快速冷冻体外培养发育率为73.3％(33／45)，切割取样牛胚胎在25％甘油+0.25M蔗糖+20％血清的PBS为冷冻液快速冷冻后移植妊娠率为57.1％(4／7)。
4．依据牛Y染色体特异序列设计合成5对Y染色体特异引物，依据牛骨胳肌α肌动蛋白前体基冈和微卫星DNA序列设计合成4对牛DNA特异内标引物。应用合成的引物单重PCR扩增牛血样DNA，筛选到4对牛Y染色体特异引物和一对牛DNA特异内标引物。应用所筛选的4对牛Y—染色体特异引物和内标引物分别PCR扩增牛基因组DNA、成纤维细胞和胚胎，筛选出2个可用于牛胚胎性别鉴别的多重PCR引物组合：B34／A12、B78／A12。建立了两温度循环PCR方法(94℃变性15s和55℃退火15S，以94℃变性1s和55℃退火1s)并鉴别牛胚胎性别，将PCR扩增时间减少到50分钟左右。
5．设计合成的5对牛Y—染色体特异引物都可以扩增牛和牦牛Y—染色体，引物B90在绵羊中也有扩增产物，其他动物均未获得扩增产物。扩增产物序列测定结果表明，2对牛Y—染色体特异引物(引物B90、B12)所扩增牦牛的产物序列为已知序列，2对牛Y—染色体特异引物(引物B78、B34)所扩增牦牛的产物序列为新序列。
The splitting and embryo cells sampling techniques of mouse and bovine pre-implantation embryos, the cryo-preservation of split-sampled mouse and bovine embryos , and the sexing of pre-implantation bovine embryos by PCR were studied systematicly. Genomic DNA of six farm animal species such as cattle, yak, goat, sheep, mouse and pig were amplificated by PCR with bovine Y-chromosome specific primers in this study, and the conservative sequences in different species were cloned and sequenced. The results were as follows:
1. Simple technique of mouse and cattle embryos splitting and embryo cells(5-10cells) sampling under stereo-microscope with glass needle or metal razor blade. was established. Development rates of split-sampled mouse and bovine embryos culture in vitro for 48 hours were 76.7%(46/60) and 80%(16/20), respectively. The pregnant rate of recipients of split -sampled bovine fresh and frozen-thawed embryos after transfer were 47.1% and 42.9%, respectively.
2. The total development rates of split-sampled mouse embryos in vitro after frozen-thawed by traditional freezing method in 10% Glycerol, 10% ethylene glycol, 10% glycerol + 10% dextran + 0.1M sucrose and 10% ethylene glycol+ 10% dextran + 0.1M sucrose freezing solution were 56.9%(259/455), 81.8% (317/), 84.8% (540/738) and 59.5% (308/518) , respectively. Short time (0-4hours ) in vitro culture of splitted angd sampled mouse embryos before freezing did not significantly effect on their development rate after frozen-thawed..
3. Quick-freezing technique of split-sampled mouse and bovine embryos was established. The total development rate of quick-freezing split-sampled mouse embryos culture 48 hours in vitro was 73.3% (33/45) ,. The rate of pregnant recipients of frozen-thawed split-sampled bovine embryos, frozen in 25% Glycerol + 0.25M sucrose +20%serum PBS, was 57.1%
(4/7) .
4. Five pairs of bovine male-specific PCR primers were designed based on the sequences of bovine Y-specific repeat sequence, testis-specific protein gene from Y-chromosome (TSPY) and sex determining region Y-protein gene (SRY) from GenBank. Meanwhile, based on skeletal alpha action precursor gene and Bovine 1.715 satellite DNA, 4 pairs of bovine-specific PCR primers were designed as internal control primers. After PCR
引文
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