CN111534479A - 一种提高牛体外受精胚和克隆胚的发育质量的培养液 - Google Patents
一种提高牛体外受精胚和克隆胚的发育质量的培养液 Download PDFInfo
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Abstract
本发明公开了一种提高牛体外受精胚和克隆胚的发育质量的培养液,属于家畜胚胎工程技术领域。本发明公开的一种提高牛体外受精胚和克隆胚的发育质量的培养液,包括SOF基础培养基,还包括以下添加剂:孕酮、维生素E、L‑肉碱、FGF、皮质酮、瓜氨酸、EGF、IGF和LIF。本发明公开的一种提高牛体外受精胚和克隆胚的发育质量的培养液,可有效提高牛体外受精胚和体细胞克隆胚胎的发育率和发育质量。
Description
技术领域
本发明涉及家畜胚胎工程技术领域,更具体的说是涉及一种提高牛体外受精胚和克隆胚的发育质量的培养液。
背景技术
家畜胚胎体外培养是一项具有潜在的研究价值和商用价值的技术,可广泛应用于高遗传价值牛个体的培育,高遗传价值牛群的培育及动物基因编辑育种。但至今体外培养获得的胚胎在质量及数量上往往不及在体内正常发育的胚胎,体外获得的胚胎在胚胎致密化阶段发育迟缓,存在氧化应激和生长能力不足的缺陷,导致胚胎卵裂率逐渐下降,形成囊胚的细胞数目少,进而影响胚胎质量,制约了早期胚胎体外发育率,显著制约着此技术的广泛应用。
因此,提供一种提高牛体外受精胚和克隆胚的发育质量的培养液是本领域技术人员亟需解决的问题。
发明内容
有鉴于此,本发明提供了一种提高牛体外受精胚和克隆胚的发育质量的培养液,可有效提高牛体外受精胚和体细胞克隆胚胎的发育率和发育质量。
为了实现上述目的,本发明采用如下技术方案:
一种提高牛体外受精胚和克隆胚的发育质量的培养液,包括SOF基础培养基,所述培养液还包括以下添加剂:瓜氨酸、孕酮、维生素E、L-肉碱、FGF、皮质酮、EGF、IGF和LIF。
孕酮和皮质酮能够较好地维持胚胎细胞的体外存活和增殖;维生素E能限制ROS的有害作用;L-肉碱能促进胚胎内部脂肪代谢产生能量;瓜氨酸能够调控细胞内NO信号,维持氮稳定;FGF,EGF,IGF和LIF都具有促进细胞生长增殖,改善细胞形态提高细胞活力的功能。
九种添加剂添加到培养液中,可有效降低牛体外受精胚的氧化损伤,提高牛体外受精胚发育率和发育质量。
进一步,所述孕酮的浓度为1-100ng/mL,维生素E的浓度为10-1000μM,L-肉碱的浓度为0.1-30mM,FGF的浓度为1-100ng/mL,皮质酮的浓度为1-100ng/mL,EGF的浓度为1-100ng/mL,IGF的浓度为1-100ng/mL,LIF的浓度为1-100ng/mL。
优选地,孕酮的浓度为1-10ng/mL,维生素E的浓度为10-100μM,L-肉碱的浓度为1-3mM,FGF的浓度为10-100ng/mL,皮质酮的浓度为1-10ng/mL,瓜氨酸的浓度为0.01-1mg/mL,EGF的浓度为1-10ng/mL,IGF的浓度为10-100ng/mL,LIF的浓度为10-100ng/mL。
进一步,所述培养液还包括必需氨基酸、非必需氨基酸、无脂肪酸牛血清白蛋白、胰岛素-转铁蛋白-硒添加剂、β-巯基乙醇、亚牛磺酸、1-油酰基-sn-甘油-3-磷酸钠盐和红景天苷。
胰岛素-转铁蛋白-硒添加剂、β-巯基乙醇、亚牛磺酸、1-油酰基-sn-甘油-3-磷酸钠盐和红景天苷于胚胎发育之前添加到培养体系中,能够充分发挥其作为胚胎培养环境作用因子的积极作用,高效率地获得牛体外受精胚胎,显著提高牛体外受精胚胎发育率、囊胚数及囊胚细胞数,大幅降低制备胚胎的生产成本。
进一步,所述必需氨基酸体积分数为0.1-2%,非必需氨基酸体积分数为0.1-2%,无脂肪酸牛血清白蛋白浓度为1-10mg/mL,胰岛素-转铁蛋白-硒添加剂体积分数为0.1-2%,β-巯基乙醇浓度为10-100μM,亚牛磺酸浓度为1-10mM,1-油酰基-sn-甘油-3-磷酸钠盐浓度为0.1-1μg/mL,红景天苷浓度为0.01-0.2mM。
通过控制必需氨基酸、非必需氨基酸及无脂肪酸牛血清白蛋白可进一步提高牛胚胎体外发育质量。
进一步,每100mL所述SOF基础培养基包括26.168mg CaCl2·2H2O、9.999mgC6H5Na3O7·2H2O、2.923mg谷氨酰胺、37.218mg MgSO4·7H2O、49.904mg肌醇、1.000mg酚红、53.378mg KCl、16.195mg KH2PO4、4.402mg丙酮酸钠、210.025mg NaHCO3、629.399mg NaCl和0.0757mg乳酸钠。
进一步,所述培养液还包括12μg/mL庆大霉素。
进一步,一种提高牛体外受精胚和克隆胚的发育质量的培养方法,将牛体外受精胚或者克隆胚置于上述的培养液中进行培养。
进一步,一种提高牛体外受精胚和克隆胚的发育质量的培养方法,将所述培养液预热至少2小时,将牛精卵结合12-16h的体外受精胚或在6-DMAP中完成化学激活的体细胞克隆胚加入所述培养液中,CO2培养箱中培养7-8天。
进一步,一种提高牛体外受精胚和克隆胚的发育质量的培养方法,将所述体外受精胚或者所述体细胞克隆胚在38.5℃、5%CO2、7%O2饱和湿度下培养。
经由上述的技术方案可知,与现有技术相比,本发明公开提供了一种提高牛体外受精胚和克隆胚的发育质量的培养液,可有效提高牛体外受精胚和体细胞克隆胚胎的发育率和发育质量。
具体实施方式
下面将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。
胰蛋白酶、庆大霉素、头孢霉素、无机盐、石蜡油(M8410)为sigma公司产品;
无Hepes的M199基础培养液为Thermo公司产品;
特级胎牛血清(FBS)为Gibco公司产品;
必需氨基酸为Thermo Fisher公司产品(货号1831512);
非必需氨基酸为Thermo Fisher公司产品(货号1958909);
无脂肪酸牛血清白蛋白EFAF-BSA为sigma公司产品(货号A6003-5G);
胰岛素-转铁蛋白-硒添加剂ITS-G为Thermo Fisher公司产品(货号1865342,胰岛素1g/L,转铁蛋白0.55g/L,硒0.00067g/L);
β-巯基乙醇为Thermo Fisher公司产品(货号1922445);
亚牛磺酸为sigma公司产品(货号H1384);
1-油酰基-sn-甘油-3-磷酸钠为Enzo Life Science公司产品(货号LP100-0025);
红景天苷为sigma公司产品(货号SMB00072-1MG);
SOF基础培养基为自行配制;
其他未标明的均为sigma公司产品。
采卵液(PBS液):26.168mg CaCl2·2H2O、37.218mg MgSO4·7H2O、0.0203g KCl、0.0200g KH2PO4、0.0036g丙酮酸钠、0.805gNaCl、0.1153g Na2HPO4、D-Glucose 0.1g、肝素钠6mg、胎牛血清3mL,去离子水定容到100mL,1mM NaOH调pH到7.0-7.2。
卵母细胞体外成熟培养液:无Hepes的M199基础培养液中添加6mg/mL无脂肪酸牛血清白蛋白、0.075IU/mL HMG、2μg/mL 17β-雌二醇、60ng/mL EGF、40ng/mL bFGF和50ng/mLCXCL12、50μg/mL叶酸、2μg/mL胆酸。
SOF溶液:26.168mg CaCl2·2H2O、9.999mg C6H5Na3O7·2H2O、2.923mg谷氨酰胺、37.218mg MgSO4·7H2O、49.904mg肌醇、1.000mg酚红、53.378mg KCl、16.195mg KH2PO4、4.402mg丙酮酸钠、210.025mg NaHCO3、629.399mg NaCl和0.0757mg乳酸钠,去离子水定容到100mL,1mM NaOH调pH到7.2-7.4。
SOFaa溶液:以SOF溶液作为基础培养液,添加体积分数1%的必需氨基酸、体积分数1%的非必需氨基酸、6mg/mL无脂肪酸牛血清白蛋白、12g/mL庆大霉素。
mSOFaa溶液(本发明提高牛体外受精胚和克隆胚的发育质量的培养液):以SOFaa溶液作为基础培养液,预热时添加体积分数为1%的ITS-G、55μMβ-巯基乙醇、2.5mM亚牛磺酸、0.4μg/mL 1-油酰基-SN-甘油-3-磷酸钠盐、0.1mM红景天苷、10ng/mL孕酮、100μM维生素E、1mM L-肉碱、0.1mg/mL瓜氨酸40ng/mL FGF、10ng/mL皮质酮、20ng/mL EGF、25ng/mL IGF、20ng/mL LIF。
受精液:以不含肌醇的SOF溶液作为基础培养液,还包含有48mg/mL无脂肪酸牛血清白蛋白、12μg/mL庆大霉素、63μg/mL L-精氨酸、6μg/mL L-天冬氨酸、3mM L-肉碱、2mM肾上腺素、20mM青霉胺、10mM亚牛磺酸、10μg/mL肝素。
胚胎移植液:以SOFaa溶液作为基础培养液,预热时添加体积分数为1%的ITS-G、100μMβ-巯基乙醇、2.5mM亚牛磺酸、0.4μg/mL 1-油酰基-sn-甘油-3-磷酸钠盐、0.1mM红景天苷、0.04mg/mL的L-瓜氨酸、2.38mg/mL 4-羟乙基哌嗪乙磺酸、0.168mg/mL碳酸氢钠。
实施例1牛体外受精胚胎的制备
(1)卵母细胞的成熟培养
牛卵子采自陕西省杨凌科元克隆股份有限公司的奶牛(采集时间2019年1月至2019年12月),使用活体采卵仪将注射过FSH牛体内的卵巢上的卵泡中的卵子采集并置于装有无菌的38.5℃的采卵液中,0.5h以内运回实验室。卵子取回后,在实体显微镜下收集卵丘-卵母细胞复合体(cumulus-oocyte complexes,COCs);收集后在采卵液中清洗三遍。选择卵母细胞形态正常且颗粒细胞完整的的COCs用于体外成熟培养(记为总卵母细胞数)。
将选择的COCs在卵母细胞体外成熟培养液中洗两遍,然后移入装有500μL卵母细胞体外成熟培养液(提前在38.5℃培养箱平衡一小时)的四孔板中,在38.5℃、5%CO2、饱和湿度条件下培养20-22h。将培养成熟的COCs用1000mL移液枪反复吹打,以除去卵母细胞外扩散的卵丘细胞。吹打至COCs周围只剩下紧密包裹的四到五层卵丘细胞时,在受精液(BO液)中洗2次,洗干净吹下的游离卵丘细胞,然后将紧密包裹四到五层卵丘细胞的卵母细胞移至受精液滴(提前放置到38.5℃培养箱中平衡2h以上)中。
(2)精子的解冻、纯化、受精
将冻精(西安市奶牛中心,2013年10月采集)从液氮罐取出后,放置于37℃水浴解冻,离心管中做Percoll分层液。将2mL 90%Percoll液置于离心管底部,把2mL 45%Percoll液小心加到90%Percoll液面上,再将解冻后的精子置于45%Percoll液面上,1500rpm离心20min,小心吸取底部约200μL精液加入至含有卵母细胞的受精液中,放回培养箱内使精卵进行结合20h。
(3)受精胚胎的培养
处理组:于四孔板中添加mSOFaa溶液(本发明提高牛体外受精胚和克隆胚的发育质量的培养液)、2mg/mL透明质酸,每孔500μL,液面上覆盖500μL石蜡油,预先在38.5℃、5%CO2、饱和湿度培养箱中平衡至少2h。
受精完毕后用口吸管或者枪头吹去卵子周围的残存卵丘细胞和精子,获得假定受精的牛体外受精胚,置于四孔板的mSOFaa溶液中,每孔40枚左右,38.5℃、5%CO2、7%O2饱和湿度培养箱中培养72h,挑出未卵裂的受精胚,放回培养箱继续培养至第七天(168h),并挑选形态正常的囊胚胚胎装入含胚胎移植液的胚胎移植管中进行胚胎移植。在移植后的第33~38天用B超检查受体牛是否怀孕。
对照组用SOFaa溶液培养,其余操作与处理组一致。
观察并记录各组发育情况,试验结果见表1。
表1
注:a、b上标不同表示差异显著,显著性使用卡方检验来检测。
从表1的实验结果可以看出,使用本发明的培养液进行胚胎培养,得到的囊胚比例更多;说明本发明的培养液能提高现有体外培养阶段的胚胎生产效率。
胚胎移植后,统计怀孕率,结果见表2。
表2
注:a、b上标不同表示差异显著,显著性使用卡方检验来检测。
从表2的实验结果可以看出,使用本发明的培养液进行胚胎培养,得到的囊胚在移植后怀孕的牛比例更高。结合表1,说明本发明的培养液能提高现有的总体外胚胎生产效率。
实施例2牛体细胞克隆胚胎的制备
(1)牛胎儿成纤维细胞的培养
从液氮中取一管荷斯坦奶牛的2~5代的牛胎儿成纤维细胞(采集自杨凌科元克隆股份有限公司牛场)于39℃解冻,加0.8mL的DMEM/F12细胞培养液,离心,弃上清,加细胞培养液重悬,取3mL细胞悬浮液接种于直径6cm的培养皿中,置于CO2培养箱中38.5℃条件下培养。
待牛胎儿成纤维细胞达到80%汇合时,吸弃培养液,用无Ca2+、Mg2+的PBS冲洗细胞,加入胰蛋白酶与EDTA混合消化液,消化细胞。在倒置显微镜下观察细胞,待大多数细胞回缩、变圆、细胞间隙扩大时,用含10%胎牛血清的DMEM/F12细胞培养液终止消化,用移液器吹打后,离心收集,悬浮,按1:3的比例接种于24孔板,放入CO2培养箱中培养。进行体细胞克隆胚制作的前两天,将培养液更换成含0.5%胎牛血清的DMEM/F12。
(2)卵母细胞的成熟培养
将选择的COCs在牛卵母细胞体外成熟培养液中洗两遍,然后移入装有3mL的体外成熟培养液的3cm平皿中(提前在培养箱平衡一小时),在38.5℃、5%CO2、饱和湿度条件下培养18-20h。将培养成熟的COCs放入含0.1%透明质酸酶的生理盐水中消化1~2min,并用1000mL移液枪反复吹打,以除去卵母细胞外扩散的卵丘细胞。吹打干净后,在PBS中洗3次,然后在实体视显微镜下,以异物针拨动卵母细胞,挑选有极体的卵母细胞待用。
(3)牛体细胞克隆胚的构建
去核:
去核前卵母细胞放在含7.5μg/mL细胞松弛素B、4mg/mL不含脂肪酸BSA的含有Hepes的M199中,于38.5℃孵育30min。在显微操作仪下,用内径为20μm的去核管吸取第一极体及其周围的部分突出卵胞质。
注核和电融合:
注核时,挑选直径为15~20μm的牛胎儿成纤维细胞注入去核的卵母细胞透明带下。注核后的重组胚采用微电极的方法进行融合。融合前,重组胚在电融合液中预平衡3min,电融合在150倍的显微操作仪下进行。用于融合操作的两根“Z”字形微电极顶端直径为15μm,后端连于显微操作仪上,使重组胚的供体、受体细胞膜接触面与两电极的连线垂直,融合参数:电压32V、脉冲时长20μs、2次脉冲间隔10ms。融合后半小时在显微镜下观察融合情况,将融合好的胚胎放回卵母细胞体外成熟培养液中,继续培养3小时。
(4)牛体细胞克隆重组胚的激活
在电融合之后3h,将牛体细胞克隆重组胚通过离子霉素(Ionomycin)联合6-DMAP的激活处理进行激活:首先在含2~5μmol/L离子霉素的SOFaa溶液中室温孵育4min,用不含离子霉素的SOFaa洗重组胚胎3-5次,然后在含1~2mmol/L 6-DMAP的SOFaa溶液中,在38.5℃、5%CO2、饱和湿度条件下培养4h。
(5)牛体细胞克隆胚胎的体外培养
处理组:于四孔板中添加mSOFaa溶液(本发明提高牛体外受精胚和克隆胚的发育质量的培养液)、2mg/mL透明质酸,每孔加入500μL,mSOFaa溶液上还覆盖有500μL石蜡油,预先在CO2培养箱中平衡至少2h,牛体细胞克隆重组胚在进行激活处理后,转移到上述四孔板中。每孔放置35~40枚牛体细胞克隆重组胚,在38.5℃、5%CO2、7%O2及饱和湿度条件下培养。
对照组用SOFaa溶液培养,其余操作与处理组一致。
记录发育情况,结果见表3。
表3
注:a、b上标不同表示差异显著,显著性使用卡方检验来检测。AB级囊胚为可移植囊胚,C级为不推荐移植囊胚,囊胚ABC分级是根据国际胚胎移植协会的囊胚质量评分标准来评定。
从表3的实验结果可以看出,使用本发明的培养液进行胚胎培养,得到的克隆囊胚比例更多;说明本发明的培养液能提高现有体外培养阶段的克隆胚胎生产效率。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (9)
1.一种提高牛体外受精胚和克隆胚的发育质量的培养液,包括SOF基础培养基,其特征在于,所述培养液还包括以下添加剂:孕酮、维生素E、L-肉碱、FGF、皮质酮、瓜氨酸、EGF、IGF和LIF。
2.根据权利要求1所述的一种提高牛体外受精胚和克隆胚的发育质量的培养液,其特征在于,所述孕酮的浓度为1-100ng/mL,维生素E的浓度为10-1000μM,L-肉碱的浓度为0.1-30mM,FGF的浓度为1-100ng/mL,皮质酮的浓度为1-100ng/mL,瓜氨酸的浓度为0.01-1mg/mL,EGF的浓度为1-100ng/mL,IGF的浓度为1-100ng/mL,LIF的浓度为1-100ng/mL。
3.根据权利要求1或2所述的一种提高牛体外受精胚和克隆胚的发育质量的培养液,其特征在于,所述培养液还包括必需氨基酸、非必需氨基酸、无脂肪酸牛血清白蛋白、胰岛素-转铁蛋白-硒添加剂、β-巯基乙醇、亚牛磺酸、1-油酰基-sn-甘油-3-磷酸钠盐和红景天苷。
4.根据权利要求3所述的一种提高牛体外受精胚和克隆胚的发育质量的培养液,其特征在于,所述必需氨基酸体积分数为0.1-2%,非必需氨基酸体积分数为0.1-2%,无脂肪酸牛血清白蛋白浓度为1-10mg/mL,胰岛素-转铁蛋白-硒添加剂体积分数为0.1-2%,β-巯基乙醇浓度为10-100μM,亚牛磺酸浓度为1-10mM,1-油酰基-sn-甘油-3-磷酸钠盐浓度为0.1-1μg/mL,红景天苷浓度为0.01-0.2mM。
5.根据权利要求4所述的一种提高牛体外受精胚和克隆胚的发育质量的培养液,其特征在于,每100mL所述SOF基础培养基包括26.168mg CaCl2·2H2O、9.999mg C6H5Na3O7·2H2O、2.923mg谷氨酰胺、37.218mg MgSO4·7H2O、49.904mg肌醇、1.000mg酚红、53.378mgKCl、16.195mg KH2PO4、4.402mg丙酮酸钠、210.025mg NaHCO3、629.399mg NaCl和0.0757mg乳酸钠。
6.根据权利要求5所述的一种提高牛体外受精胚和克隆胚的发育质量的培养液,其特征在于,所述培养液还包括12μg/mL庆大霉素。
7.一种提高牛体外受精胚和克隆胚的发育质量的培养方法,其特征在于,将牛体外受精胚或者克隆胚置于权利要求1-6中任一项所述的培养液中进行培养。
8.根据权利要求7所述的一种提高牛体外受精胚和克隆胚的发育质量的培养方法,其特征在于,将所述培养液预热至少2小时,将牛精卵结合12-16h的体外受精胚或在6-DMAP中完成化学激活的体细胞克隆胚加入所述培养液中,CO2培养箱中培养7-8天。
9.根据权利要求8所述的一种提高牛体外受精胚和克隆胚的发育质量的培养方法,其特征在于,将所述体外受精胚或者所述体细胞克隆胚在38.5℃、5%CO2、7%O2饱和湿度下培养。
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