CN110205299A - 一种新型鸡cef重编程为pgc的诱导体系的建立方法 - Google Patents

一种新型鸡cef重编程为pgc的诱导体系的建立方法 Download PDF

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CN110205299A
CN110205299A CN201910514421.9A CN201910514421A CN110205299A CN 110205299 A CN110205299 A CN 110205299A CN 201910514421 A CN201910514421 A CN 201910514421A CN 110205299 A CN110205299 A CN 110205299A
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李碧春
金晶
左其生
赵瑞丰
李婷婷
袁霞
靳锴
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Yangzhou University
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Abstract

本发明涉及一种新型鸡CEF重编程为PGC的诱导体系的建立方法,属于生物技术领域。在分离的原代CEF中,慢病毒外源导入OCT4,SOX2,NANOG,LIN28重编程因子,连续培养21天后,利用SSEA‑1抗体进行iPSC细胞株鉴定,鉴定为阳性的细胞株进行更换为BMP4/BMP8b/EGF诱导体系,诱导成为iPGC。本发明解决了濒危品种鸡PGC获取的数量低的问题,实现了濒危鸡品种的种质资源保护和物种复原工作。

Description

一种新型鸡CEF重编程为PGC的诱导体系的建立方法
技术领域
本发明涉及一种新型鸡CEF重编程为PGC的诱导体系的建立方法,属于生物技术领域。
背景技术
在哺乳动物家畜中,体细胞核移植、体外受精和胚胎移植技术均已成功,能够结合精子、卵子、胚胎甚至是体细胞的冻存,实现种质资源的保护和物种复原。但是由于卵生的特点,上述方法均难以在家禽上实现。
家禽的原始生殖细胞(Primordial germ cell, PGC)具有独特的通过血液迁移定居于性腺的特点,使得PGC迁移路径成为实现禽类物种复原的唯一途径。但是单个胚胎所获取的PGC远不足以满足实际应用所需。尤其是濒危品种的PGC来源成为了限制这一技术的难题。而禽类的成纤维细胞(CEF)能够在短期内大量获取,若能将其诱导成PGC无疑是解决禽类物种复原的最佳路径。
发明内容
本发明旨在克服禽类体细胞诱导形成生殖细胞的障碍难题,提供一种重复性好又高效的鸡CEF重编程为PGC的诱导体系,即一种新型鸡CEF重编程为PGC的诱导体系的建立方法。
本发明的技术方案如下:
一种新型鸡CEF重编程为PGC的诱导体系的建立方法,其特征是,在分离好的原代CEF中,慢病毒外源导入OCT4, SOX2, NANOG, LIN28重编程因子,连续培养21天后,利用SSEA-1抗体进行iPSC细胞株鉴定,鉴定为阳性的细胞进行更换为BMP4/BMP8b/EGF诱导体系,诱导成为iPGC。
优选地,具体操作方法如下:
步骤1:取出10日龄的鸡胚,放入加有1%青链霉素的PBS中,去除四肢,头及内脏,剩余组织使用胰酶消化5min,吹匀,400目滤筛过滤后制备CEF细胞悬液,以1200rpm/min离心六分钟,弃上清以培养液A成悬,1×105个细胞/孔接种于24孔板中,培养24小时;
步骤2:四种慢病毒以1:1:1:1的比例混合,以感染复数MOI=10与 400μl培养液B混合,加入24孔板中培养6小时后弃去含病毒的培养液B,换入新的培养液B,18小时后换培养液A,48小时观察转染效果;
步骤3:观察结束后将培养液A更换为培养液C,培养48小时后可以观察到细胞形态由梭状逐渐变化成圆形并出现聚团和克隆生长现象;
步骤4:继续培养48小时后,可以观察到克隆逐渐明显,进行单细胞克隆挑取;在体式显微镜下通过口吸管挑取iPS样克隆集落,加入含有20μlaccutase的96孔板中消化10分钟,再将细胞悬液重新接种于铺有饲养层细胞的96孔板中,加入100μl的培养液C进行培养;
步骤5:继续培养14天直至典型iPSC细胞出现,期间每3-5天根据细胞状态更换饲养层细胞;
步骤6:将SSEA-1红色荧光直标抗体用PBS稀释100倍,每孔加入5μl稀释后抗体混匀,2小时后在倒置荧光显微镜下观察细胞是否能标记上红色荧光,筛选iPSC细胞系;
步骤7:将能标记上红色荧光的细胞由96孔板传代至铺有2×105个细胞/孔的24孔板中传代培养;
步骤8:弃去24孔板中的上清,加入200μl Accutase消化带有饲养层细胞的iPSC 10分钟;加入400μl培养液C终止消化,差速贴壁40分钟取上清以1×105个细胞/孔接种于以16.7μg/ml人胎盘膜黏连蛋白包被1小时的24孔板中,以培养液D进行培养;
步骤9:培养4天后通过Cvh抗体标记后,流式细胞分选收集阳性细胞记为iPGC。
优选地,所述步骤5中,更换饲养层细胞操作方法:按照步骤1中的方法分离CEF,以1×106个细胞/60mm皿接种以培养液A培养24小时;以含有10μg/ml丝裂霉素C的培养液A处理2.5小时后更换为普通的培养液A继续培养24小时,以5×104个细胞/孔接种96孔板。
优选地,所述培养液A由45mlDMEM高糖培养基、5%FBS、1%青链霉素构成。
优选地,所述培养液B由培养液A、5μg/ml polybrene构成。
优选地,所述培养液C由43.5ml KO-DMEM培养基、5%FBS、1%鸡血清、1%青链霉素、0.4%非必须氨基酸、0.2μlβ-巯基乙醇、100μl hSCF (5ng/ml)、100μlbfgf(10ng/ml)、50μlLIF(10ng/ml)构成。
优选地,所述培养液D由培养液C、500ng/mlBMP4、500 ng/ml BMP8b、50 ng/ml EGF构成。
本发明的有益效果如下:
本发明解决了濒危品种鸡PGC获取的数量低的问题,实现了濒危鸡品种的种质资源保护和物种复原工作。
附图说明
图1为本发明中CEF在第五天发生形态变化,出现聚团生长的细胞克隆示意图。
具体实施方式
本发明中所需原材料(括号为厂商):
金开瑞公司构建的OCT4, SOX2, NANOG, LIN28过表达载体,并慢病毒包装好,滴度为1×108UI/mL;孵化至10日龄的鸡胚。
DMEM高糖培养基(Hyclone),FBS(Gibco), 青链霉素(碧云天),Polybrene(碧云天),KO-DMEM(Gibco),鸡血清(Gibco),非必须氨基酸(sigma),β-巯基乙醇(sigma),hSCF(sigma),bfgf(sigma),Lif(sigma),BMP4(Prospec), BMP8b(R&D Systems),EGF (R&DSystems),accutase(Gibco),SSEA-1(R&D Systems),Cvh(abcam),丝裂霉素C(sigma)。
本发明中所需培养基配方为:
培养液A:DMEM高糖培养基,(体积浓度百分比)5%FBS, (体积浓度百分比)1%青链霉素。
培养液B: 培养液A,5μg/ml polybrene(聚凝胺)。
培养液C:43.5ml KO-DMEM培养基,(体积浓度百分比)5%FBS,(体积浓度百分比)1%鸡血清,(体积浓度百分比)1%青链霉素,(体积浓度百分比)0.4%非必须氨基酸,0.2μlβ-巯基乙醇,100μl hSCF (5ng/ml),100μlbfgf(10ng/ml),50μl LIF(10ng/ml)。
培养液D:培养液C,500ng/mlBMP4,500 ng/ml BMP8b, 50 ng/ml EGF。
本发明具体步骤为:
步骤1:取出10日龄的鸡胚,放入加有1%青链霉素的PBS中,去除四肢,头及内脏,剩余组织使用胰酶消化5min,吹匀,400目滤筛过滤后制备CEF细胞悬液,以1200rpm/min离心六分钟,弃上清以培养液A成悬,1×105个细胞/孔接种于24孔板中,培养24小时。
步骤2:四种慢病毒以1:1:1:1的比例混合,以感染复数MOI=10与 400μl培养液B混合,加入24孔板中培养6小时后弃去含病毒的培养液B,换入新的培养液B,18小时后换培养液A,48小时观察转染效果。
步骤3:观察结束后将培养液A更换为培养液C,培养48小时后可以观察到细胞形态由梭状逐渐变化成圆形并出现聚团和克隆生长现象(如图1所示)。
步骤4:继续培养48小时后,可以观察到克隆逐渐明显,进行单细胞克隆挑取。在体式显微镜下通过口吸管挑取iPS样克隆集落,加入含有20μlaccutase的96孔板中消化10分钟,再将细胞悬液重新接种于铺有饲养层细胞的96孔板中,加入100μl的培养液C进行培养。
步骤5:继续培养14天直至典型iPSC细胞出现,期间每3-5天根据细胞状态更换饲养层细胞。更换饲养层细胞操作方法:按照步骤1中的方法分离CEF,以1×106个细胞/60mm皿接种以培养液A培养24小时。以含有10μg/ml丝裂霉素C的培养液A处理2.5小时后更换为普通的培养液A继续培养24小时,以5×104个细胞/孔接种96孔板。
步骤6:将SSEA-1红色荧光直标抗体用PBS稀释100倍,每孔加入5μl稀释后抗体混匀,2小时后在倒置荧光显微镜下观察细胞是否能标记上红色荧光,筛选iPSC细胞系。
步骤7:将能标记上红色荧光的细胞由96孔板传代至铺有2×105个细胞/孔的24孔板中传代培养。
步骤8:弃去24孔板中的上清,加入200μl Accutase消化带有饲养层细胞的iPSC10分钟。加入400μl培养液C终止消化,差速贴壁40分钟取上清以1×105个细胞/孔接种于以16.7μg/ml人胎盘膜黏连蛋白包被的24孔板中,以培养液D进行培养。
步骤9:培养4天后通过Cvh抗体标记后,流式细胞分选收集阳性细胞记为iPGC。

Claims (7)

1.一种新型鸡CEF重编程为PGC的诱导体系的建立方法,其特征是,在分离的原代CEF中,慢病毒外源导入OCT4, SOX2, NANOG, LIN28重编程因子,连续培养21天后,利用SSEA-1抗体进行iPSC细胞株鉴定,鉴定为阳性的细胞株进行更换为BMP4/BMP8b/EGF诱导体系,诱导成为iPGC。
2.根据权利要求1所述的一种新型鸡CEF重编程为PGC的诱导体系的建立方法,其特征是,具体操作方法如下:
步骤1:取出10日龄的鸡胚,放入加有1%青链霉素的PBS中,去除四肢,头及内脏,剩余组织使用胰酶消化5min,吹匀,400目滤筛过滤后制备CEF细胞悬液,以1200rpm/min离心六分钟,弃上清以培养液A成悬,1×105个细胞/孔接种于24孔板中,培养24小时;
步骤2:四种慢病毒以1:1:1:1的比例混合,以感染复数MOI=10与 400μl培养液B混合,加入24孔板中培养6小时后弃去含病毒的培养液B,换入新的培养液B,18小时后换培养液A,48小时观察转染效果;
步骤3:观察结束后将培养液A更换为培养液C,培养48小时后可以观察到细胞形态由梭状逐渐变化成圆形并出现聚团和克隆生长现象;
步骤4:继续培养48小时后,可以观察到克隆逐渐明显,进行单细胞克隆挑取;在体式显微镜下通过口吸管挑取iPS样克隆集落,加入含有20μlaccutase的96孔板中消化10分钟,再将细胞悬液重新接种于铺有饲养层细胞的96孔板中,加入100μl的培养液C进行培养;
步骤5:继续培养14天直至典型iPSC细胞出现,期间每3-5天根据细胞状态更换饲养层细胞;
步骤6:将SSEA-1红色荧光直标抗体用PBS稀释100倍,每孔加入5μl稀释后抗体混匀,2小时后在倒置荧光显微镜下观察细胞是否能标记上红色荧光,筛选iPSC细胞系;
步骤7:将能标记上红色荧光的细胞由96孔板传代至铺有2×105个细胞/孔的24孔板中传代培养;
步骤8:弃去24孔板中的上清,加入200μl Accutase消化带有饲养层细胞的iPSC 10分钟;加入400μl培养液C终止消化,差速贴壁40分钟取上清以1×105个细胞/孔接种于以16.7μg/ml人胎盘膜黏连蛋白包被1小时的24孔板中,以培养液D进行培养;
步骤9:培养4天后通过Cvh抗体标记后,流式细胞分选收集阳性细胞记为iPGC。
3.根据权利要求2所述的一种新型鸡CEF重编程为PGC的诱导体系的建立方法,其特征是,所述步骤5中,更换饲养层细胞操作方法:按照步骤1中的方法分离CEF,以1×106个细胞/60mm皿接种以培养液A培养24小时;以含有10μg/ml丝裂霉素C的培养液A处理2.5小时后更换为普通的培养液A继续培养24小时,以5×104个细胞/孔接种96孔板。
4.根据权利要求3所述的一种新型鸡CEF重编程为PGC的诱导体系的建立方法,其特征是,所述培养液A由45mlDMEM高糖培养基、5%FBS、1%青链霉素构成。
5.根据权利要求4所述的一种新型鸡CEF重编程为PGC的诱导体系的建立方法,其特征是,所述培养液B由培养液A、5μg/ml polybrene构成。
6. 根据权利要求5所述的一种新型鸡CEF重编程为PGC的诱导体系的建立方法,其特征是,所述培养液C由43.5ml KO-DMEM培养基、5%FBS、1%鸡血清、1%青链霉素、0.4%非必须氨基酸、0.2μlβ-巯基乙醇、100μl hSCF (5ng/ml)、100μlbfgf(10ng/ml)、50μl LIF(10ng/ml)构成。
7. 根据权利要求6所述的一种新型鸡CEF重编程为PGC的诱导体系的建立方法,其特征是,所述培养液D由培养液C、500ng/mlBMP4、500 ng/ml BMP8b、50 ng/ml EGF构成。
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