CN114107180A - 无透明带体细胞克隆1-细胞期胚胎聚合及体外培养的方法 - Google Patents
无透明带体细胞克隆1-细胞期胚胎聚合及体外培养的方法 Download PDFInfo
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Abstract
本发明公开了无透明带体细胞克隆1‑细胞期胚胎聚合及体外培养的方法。采用浓度为0.3%的链霉蛋白酶溶液去除重构胚和去核卵母细胞的透明带,两两一组放入含20%牛血清的洗卵液微滴中,用矿物油覆盖约30min,将1‑细胞期的克隆胚和去核卵母细胞,用自制玻璃吸管轻轻转移至电极宽度为1mm的融合槽中1对1排列,再用自制的拨卵针轻轻将2枚卵拨贴在一起,使2枚卵胞质接触切面与电场方向垂直或近似垂直,也就是组成的“8”字卵正直于两侧电极之间,然后进行融合/聚合,将相互融合的胚胎转入自制的“U型”微滴培养体系中进行培养,体细胞克隆胚胎的卵裂率达到95.7%,囊胚发育率达到69.8%,极大改善了体外克隆重构胚的发育潜力。
Description
技术领域
本发明涉及细胞培养技术领域,具体为无透明带体细胞克隆1-细胞期胚胎聚合及体外培养的方法。
背景技术
体细胞核移植(Somatic Cell Nuclear Transfer,SCNT)是一种极其有效的动物胚胎繁殖生物技术,它能够将体细胞基因组重编程到相当于受精卵的状态,即全能性状态。这种技术可用来在去核的卵母细胞中注入一个体细胞而产生一个动物个体。自从克隆羊“Dolly”的诞生来,利用不同物种的供体细胞,克隆出了许多动物,包括小鼠、兔子、猪、牛等。
该技术在理论和实践中都具有重要的应用价值,通过胚胎细胞或者体细胞作为供核细胞来构建无数个遗传上完全相同的多个后代。这项技术在猪的研究上具有极大的发展潜力,但是猪的体细胞核移植效率仍然很低,仅为仅为1%~3%的胚胎能着床受孕,严重制约该技术的发展和实际应用。
发明内容
本发明解决的技术问题在于克服现有技术的体细胞核移植效率仍然很低缺陷,提供无透明带体细胞克隆1-细胞期胚胎聚合及体外培养的方法。所述无透明带体细胞克隆1-细胞期胚胎聚合及体外培养的方法具有提高体细胞核移植效率等特点。
为实现上述目的,本发明提供如下技术方案:无透明带体细胞克隆1-细胞期胚胎聚合及体外培养的方法,包括以下步骤,
(1)猪卵母细胞的体外成熟培养;
(2)体外重构胚的制备;
(3)体外重构胚以及去核卵母细胞透明带的去除;
(4)核移植胚胎的融合/聚合;
(5)无透明带核移植胚胎“U型”微滴培养技术。
优选的,所述步骤(1)中从当地屠宰场摘取新鲜卵巢组织立即置于盛有(28-37℃)生理盐水(含青、链霉素各20万IU/L和15万IU/L)的保温瓶内,2h内送回实验室,用适量生理盐水(28-37℃)冲洗3-5遍,盛于500mL烧杯中,放在水浴锅(37℃)里,用镊子夹取卵巢,再用生理盐水润湿灭菌纱布包裹,采用10mL注射器(16号针头)抽吸直径为3-6mm的卵泡,吸取的卵泡液汇集到置于37℃水浴保温的尖底灭菌试管中,静置15min,弃上清液,取试管底部卵泡液于表面皿中,实体显微镜下观察,捡取卵丘卵母细胞复合体(COCs),转移到DPBS液滴中,冲洗3遍,用CO2培养箱内平衡2h以上的mTCM199培养基再洗3遍,然后放在39℃,相对湿度为100%,体积分数为5%CO2培养箱中培养,在体外培养的0-22h添加10IU/mL PMSG和HCG,22-44h换液去掉激素。
优选的,所述步骤(2)中将培养成熟(38-40h)的卵母细胞用移液枪转至60mm平皿中,加入5倍体积的含1%的透明质酸酶,反复吹打直至卵丘细胞完全脱落为止,挑选排出第一极体的合格的成熟卵母细胞放入操作液(M199 Hank's)内待用,制作操作盘,然后把供体细胞连同选好的成熟卵母细胞一同转入一个长1.5cm,宽20-30mm的100μL显微操作液滴中(含7.5μg/ml CB),在倒置显微镜上,用固定吸管吸持卵母细胞,用去核针吸除第Ⅰ极体及其临近20%-30%的卵母细胞胞质,挑选圆形、表面光滑的体细胞,用注核针注入卵母细胞的卵周间隙,然后将重组胚放入含有BSA的NCSU-23培养基中培养30min,对完整的存活重组胚采用1次120V/mm的直流脉冲,60μs时程融合、激活,40min后剔除未成功融合、激活的重构胚,然后将成功融合、激活的重构胚在NCSU-23的基础液中添加7.5μg/mL CB浓度的胚胎培养液中培养4h,将重构胚转移到预先在CO2培养箱中平衡至少3h的NCSU-23液滴内39℃、5%CO2、100%湿度的培养箱中四孔培养板内,10-15h进行重构胚透明带的去除,去核卵母细胞的操作方法与重构胚的操作方法相同。
优选的,所述步骤(3)中挑选重构胚和去核卵母细胞分别放入链霉蛋白酶溶液(浓度0.3%)中,当观察到重构胚或去核卵母细胞变形时,迅速将其转移到含20%牛血清的洗卵液中洗4遍,然后两两一组(1枚重构胚和1枚去核卵母细胞)放入含20%牛血清的洗卵液微滴中,用矿物油覆盖约30min,将恢复圆形重构胚以及去核卵母细胞用玻璃吸管轻轻转移至融合电极中。
优选的,所述步骤(4)中将重构胚和去核卵母细胞分别在融合液(0.25mol/L甘露醇,0.1mmol/L氯化钙,0.1mmol/L氯化镁及0.5mmol/L HEPES)滴中洗3遍,移到融合槽中,用自制的拨卵针轻轻将2枚卵紧挨在一起,放入电极宽度为1.0mm的融合槽(BTX ECM2001电融合仪配套电激活槽)中,确保切面与电场方向垂直或近似垂直,施加交流电场(10v,5s)排序,然后采用1DC,120V/mm的直流脉冲,60μs时程进行融合、聚合,待所有胚胎融合、聚合完成后进行培养。
优选的,所述步骤(5)中为了预防无透明带胚胎相互之间粘连,聚合后的胚胎分别移NCSU-23+4mg/mL BSA洗涤3遍,然后放入预先作好的培养液微滴中,每滴放1枚融合聚合后的胚胎,在39℃、5%CO2、100%湿度下培养20-30min,然后转入“U型”微滴培养体系中。
优选的,所述的无透明带核移植胚胎“U型”微滴培养的研制方法,包括以下步骤:
(1)自制一根带把手的钢针;
(2)采用35mm直径的细胞培养皿,用胚胎培养液做10个微滴,上、下各3个,中间4个微滴;
(3)覆盖上热孵育的石蜡油,用自制锥子在每一个微滴中旋转10-15个“U型”孔,深度约为300-500μm,直径300μm左右,“U型”孔制作完成后用细玻璃针轻轻搅动各个“U型”孔使其液体充分沁入到孔内。
优选的,所述的无透明带核移植胚胎“U型”微滴培养的钢针,包括把手,所述把手的一端设置有固定装置,所述固定装置上设置有钢锥,所述钢锥锥顶直径为300μm设置。
与现有技术相比,本发明的有益效果是:本发明利用体细胞核移植技术制备猪体外重构胚和去核的卵母细胞,采用浓度为0.3%的链霉蛋白酶溶液去除重构胚和去核卵母细胞的透明带,两两一组放入含20%牛血清的洗卵液微滴中,用矿物油覆盖约30min,将恢复圆形克隆胚胎用自制玻璃吸管轻轻转移至电极宽度为1mm的融合槽(BTX ECM2001电融合仪配套电激活槽)中,用自制的拨卵针轻轻将2枚卵紧挨在一起,并使2枚卵胞质接触切面与电场方向垂直或近似垂直,然后施加交流电场(10v,5s)进行排序,在施加1次脉冲,60μs时程,120V电压进行融合/聚合。将相互粘连一起的两枚胚胎转入自制的“U型”微滴培养体系中进行培养统计卵裂率及囊胚率,猪无透明带重构胚的卵裂率达到95.7%,囊胚发育率达到69.8%,极大改善了体外重构胚的发育效率。
附图说明
图1为本发明技术流程图;
图2为本发明10X体式显微镜下体外成熟卵母细胞图;
图3为本发明体细胞核移植去核操作图;
图4为本发明体细胞核移植注射操作图;
图5为本发明重构胚和去核卵母细胞透明带去除中图;
图6为本发明重构胚和去核卵母细胞透明带去除后图;
图7为本发明重构胚和去核卵母细胞透明带去除后恢复图;
图8为本发明核移植胚胎两两融合图;
图9为本发明钢针结构示意图。
图中标号:1、把手;2、固定装置;3、钢锥。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
请参阅图1-9,本发明提供一种技术方案:无透明带体细胞克隆1-细胞期胚胎聚合及体外培养的方法,包括以下步骤:
(1)猪卵母细胞的体外成熟培养,从当地屠宰场摘取新鲜卵巢组织立即置于盛有28-37℃生理盐水(含青、链霉素各20万IU/L和15万IU/L)的保温瓶内,2h内送回实验室,用适量生理盐水(28-37℃)冲洗3-5遍,盛于500mL烧杯中,放在水浴锅(37℃)里,用镊子夹取卵巢,再用生理盐水润湿灭菌纱布包裹,采用10mL注射器(16号针头)抽吸直径为3-6mm的卵泡,吸取的卵泡液汇集到置于37℃水浴保温的尖底灭菌试管中,静置15min,弃上清液,取试管底部卵泡液于表面皿中,实体显微镜下观察,捡取卵丘卵母细胞复合体(COCs),转移到DPBS液滴中,冲洗3遍,用CO2培养箱内平衡2h以上的mTCM199培养基再洗3遍,然后放在39℃,相对湿度为100%,体积分数为5%CO2培养箱中培养,在体外培养的0-22h添加10IU/mLPMSG和HCG,22-44h换液去除激素;
(2)体外重构胚的制备,将培养成熟(38-40h)的卵母细胞用移液枪转至60mm平皿中,加入5倍体积的含1%的透明质酸酶,反复吹打直至卵丘细胞完全脱落为止,挑选排出第一极体的合格的成熟卵母细胞放入操作液(M199 Hanks)内待用,制作操作盘,然后把供体细胞连同选好的成熟卵母细胞一同转入一个长1.5cm,宽20-30mm的100μL显微操作液滴中(含7.5μg/ml CB),在倒置显微镜上,用固定吸管吸持卵母细胞,用去核针吸除第Ⅰ极体及其临近20%-30%的卵母细胞胞质,挑选圆形、表面光滑的体细胞,用注核针注入卵母细胞的卵周间隙,然后将重组胚放入含有BSA的NCSU-23培养基中培养30min,对完整的存活重组胚采用1次120V/mm的直流脉冲,60μs时程融合、激活,40min后剔除未成功融合、激活的重构胚,然后将成功融合、激活的重构胚在NCSU-23的基础液中添加7.5μg/mL CB浓度的胚胎培养液中培养4h,将重构胚转移到预先在CO2培养箱中平衡至少3h的NCSU-23液滴内39℃、5%CO2、100%湿度的培养箱中四孔培养板内,10-15h进行重构胚透明带的去除,去核卵母细胞的操作方法与重构胚的操作方法相同。
(3)体外重构胚以及去核卵母细胞透明带的去除,挑选重构胚和去核卵母细胞分别放入链霉蛋白酶溶液(浓度0.3%)中,当观察到重构胚或去核卵母细胞变形时,迅速将其转移到含20%牛血清的洗卵液中洗4遍,然后两两一组(一枚重构胚和一枚去核卵母细胞)放入含20%牛血清的洗卵液微滴中,用矿物油覆盖约30min,将恢复圆形重构胚以及去核卵母细胞用玻璃吸管轻轻转移至融合电极中;
(4)核移植胚胎的融合/聚合,将重构胚和去核卵母细胞分别在融合液(0.25mol/L甘露醇,0.1mmol/L氯化钙,0.1mmol/L氯化镁及0.5mmol/L HEPES)滴中洗3遍,移到融合槽中,用自制的拨卵针轻轻将2枚卵紧挨在一起,放入电极宽度为1mm的融合槽(BTX ECM2001电融合仪配套电激活槽)中,确保切面与电场方向垂直或近似垂直,施加交流电场(10v,5s)排序,然后采用1DC,120V/mm的直流脉冲,60μs时程进行融合、聚合,待所有胚胎融合、聚合完成后进行培养;
(5)、无透明带核移植胚胎“U型”微滴培养技术,为了预防无透明带胚胎相互之间粘连,聚合后的胚胎分别移NCSU-23+4mg/mL BSA洗涤3遍,然后放入预先作好的培养液微滴中,每滴放1枚融合聚合后的胚胎,在39℃、5%CO2、100%湿度下培养20-30min,然后转入“U型”微滴培养体系中,具体研制方法如下:
(1)自制一根带把手的钢针,包括把手1,所述把手1的一端设置有固定装置2,所述固定装置2上设置有钢锥3,所述钢锥3锥顶直径为300μm设置;
(2)采用35mm直径的细胞培养皿,用胚胎培养液做10个微滴,上、下各3个,中间4个微滴;
(3)覆盖上热孵育的石蜡油,用自制锥子在每一个微滴中旋转10-15个“U型”孔,深度约为300-500μm,直径300μm左右,“U型”孔制作完成后用细玻璃针轻轻搅动各个“U型”孔使其液体充分沁入到孔内。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
Claims (8)
1.无透明带体细胞克隆1-细胞期胚胎聚合及体外培养的方法,包括以下步骤,其特征在于:
(1)猪卵母细胞的体外成熟培养;
(2)体外重构胚的制备;
(3)体外重构胚以及去核卵母细胞透明带的去除;
(4)核移植胚胎的融合/聚合;
(5)无透明带核移植胚胎“U型”微滴培养技术。
2.根据权利要求1所述的无透明带体细胞克隆1-细胞期胚胎聚合及体外培养的方法,其特征在于:所述步骤(1)中从当地屠宰场摘取新鲜卵巢组织立即置于盛有(28-37℃)生理盐水(含青、链霉素各20万IU/L和15万IU/L)的保温瓶内,2h内送回实验室;用适量生理盐水(28-37℃)冲洗3-5遍,盛于500mL烧杯中,放在水浴锅(37℃)里;用镊子夹取卵巢,再用生理盐水润湿灭菌纱布包裹,采用10mL注射器(16号针头)抽吸直径为3-6mm的卵泡,吸取的卵泡液汇集到置于37℃水浴保温的尖底灭菌试管中,静置15min,弃上清液;取试管底部卵泡液于表面皿中,实体显微镜下观察,捡取卵丘卵母细胞复合体(COCs),转移到DPBS液滴中,冲洗3遍;用CO2培养箱内平衡2h以上的mTCM199培养基再洗3遍,然后放在39℃,相对湿度为100%,体积分数为5%CO2培养箱中培养,在体外培养的0-22h添加10IU/mL PMSG和HCG,22-44h换液去掉激素。
3.根据权利要求1所述的无透明带体细胞克隆1-细胞期胚胎聚合及体外培养的方法,其特征在于:所述步骤(2)中将培养成熟(38-40h)的卵母细胞用移液枪转至60mm平皿中,加入5倍体积的含1%的透明质酸酶,反复吹打直至卵丘细胞完全脱落为止,挑选排出第一极体的合格的成熟卵母细胞放入操作液(M199 Hank’s)内待用,制作操作盘,然后把供体细胞连同选好的成熟卵母细胞一同转入一个长1.5cm,宽20-30mm的100μL显微操作液滴中(含7.5μg/ml CB),在倒置显微镜上,用固定吸管吸持卵母细胞,用去核针吸除第Ⅰ极体及其临近20%-30%的卵母细胞胞质,挑选圆形、表面光滑的体细胞,用注核针注入卵母细胞的卵周间隙,然后将重组胚放入含有BSA的NCSU-23培养基中培养30min,对完整的存活重组胚采用1次120V/mm的直流脉冲,60μs时程融合、激活,40min后剔除未成功融合、激活的重构胚,然后将成功融合、激活的重构胚在NCSU-23的基础液中添加7.5μg/mL CB浓度的胚胎培养液中培养4h,将重构胚转移到预先在CO2培养箱中平衡至少3h的NCSU-23液滴内39℃、5%CO2、100%湿度的培养箱中四孔培养板内,10-15h进行重构胚透明带的去除,去核卵母细胞的操作方法与重构胚的操作方法相同。
4.根据权利要求1所述的无透明带体细胞克隆1-细胞期胚胎聚合及体外培养的方法,其特征在于:所述步骤(3)中挑选重构胚和去核卵母细胞分别放入链霉蛋白酶溶液(浓度0.3%)中,当观察到重构胚或去核卵母细胞变形时,迅速将其转移到含20%牛血清的洗卵液中洗4遍,然后两两一组(一枚重构胚和一枚去核卵母细胞)放入含20%牛血清的洗卵液微滴中,用矿物油覆盖约30min,将恢复圆形重构胚以及去核卵母细胞用玻璃吸管轻轻转移至融合电极中。
5.根据权利要求1所述的无透明带体细胞克隆1-细胞期胚胎聚合及体外培养的方法,其特征在于:所述步骤(4)中将重构胚和去核卵母细胞分别在融合液(0.25mol/L甘露醇,0.1mmol/L氯化钙,0.1mmol/L氯化镁及0.5mmol/L HEPES)滴中洗3遍,移到融合槽中,用自制的拨卵针轻轻将2枚卵紧挨在一起,放入电极宽度为1mm的融合槽(BTX ECM2001电融合仪配套电激活槽)中,确保切面与电场方向垂直或近似垂直,施加交流电场(10v,5s)排序,然后采用1DC,120V/mm的直流脉冲,60μs时程进行融合、聚合,待所有胚胎融合、聚合完成后进行培养。
6.根据权利要求1所述的无透明带体细胞克隆1-细胞期胚胎聚合及体外培养的方法,其特征在于:所述步骤(5)中为了预防无透明带胚胎相互之间粘连,聚合后的胚胎分别移NCSU-23+4mg/mL BSA洗涤3遍,然后放入预先作好的培养液微滴中,每滴放1枚融合聚合后的胚胎,在39℃、5%CO2、100%湿度下培养20-30min,然后转入“U型”微滴培养体系中。
7.根据权利要求6所述的无透明带核移植胚胎“U型”微滴培养的研制方法,包括以下步骤,其特征在于:
(1)自制一根带把手的钢针;
(2)采用35mm直径的细胞培养皿,用胚胎培养液做10个微滴,上、下各3个,中间4个微滴;
(3)覆盖上热孵育的石蜡油,用自制锥子在每一个微滴中旋转10-15个“U型”孔,深度约为300-500μm,直径300μm左右。
8.根据权利要求7所述的无透明带核移植胚胎“U型”微滴培养的钢针,包括把手(1),其特征在于:所述把手(1)的一端设置有固定装置(2),所述固定装置(2)上设置有钢锥(3),所述钢锥(3)锥顶直径为300μm设置。
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