CN112553258A - 一种鸡成纤维细胞转分化为原始生殖细胞介导生产转基因鸡的方法 - Google Patents
一种鸡成纤维细胞转分化为原始生殖细胞介导生产转基因鸡的方法 Download PDFInfo
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Abstract
一种鸡成纤维细胞转分化为原始生殖细胞介导生产转基因鸡的方法，属于生物技术领域。通过OCT4,SOX2,NANOG,LIN28A诱导鸡CEF重编程为iPS；通过BMP4/BMP8b/EGF诱导鸡iPS分化为iPGC；鸡iPGC血管注射介导转基因鸡生产。本发明的实验方法不仅在家禽上种质资源保护过程中可行，在脊椎动物的其他研究领域也可应用。这一发明将为体外诱导的PGC‑like细胞介导后代鸡的生产研究提供更多的理论基础和技术支撑，也有利于对禽类种质资源进行保护和利用，同时也有利于转基因鸡的生产研究。
Description
技术领域
本发明涉及一种鸡成纤维细胞转分化为原始生殖细胞介导生产转基因鸡的方法，属于生物技术领域。
背景技术
PGCs移植技术是通过生殖细胞保存实现物种恢复的有效途径，Chuma S等将小鼠的PGCs移植进出生后小鼠的睾丸中使移植的PGCs分化为了精原干细胞并得到了后代。鸟类上由于PGCs能够通过血液迁移的特点，Tagami T等将白来航鸡PGCs注射至受体后成功获得后代，成功复原了隐性白羽鸡，陆阳清等通过相同的方法在白来航鸡中成功实现东兰乌鸡和白来航鸡的PGCs移植，并产生纯种黑羽鸡。PGCs由于其生殖细胞的发育潜能，能够作为种质资源进行保护和利用，也可以作为基因编辑的目的细胞生产基因编辑动物。
目前可通过重编程因子OCT4,SOX2,NANOG,LIN28A将鸡的成纤维细胞诱导重编程为诱导多能干细胞iPS，而iPS和ESCs等多能干细胞可在适当的条件下被诱导分化获得PGC-like细胞。本申请人前期也建立了BMP4/BMP8b/EGF体系，将ESCs和iPS分别诱导分化形成PGC-like细胞，同时也建立了原代PGCs迁移归巢模型且成功产生了后代。但体外诱导形成的PGC-like细胞是否具有原代PGCs种系遗传能力仍不清楚。
因此，亟需对体外转分化形成的PGC-like细胞产生后代的能力进行分析，研究通过体外鸡成纤维细胞转分化为原始生殖细胞并通过异体移植的方法产生转基因鸡的可行性和准确性，为实现PGCs在禽类资源保护过程中的广泛应用奠定基础。
发明内容
本发明提供一种新型的通过鸡成纤维细胞转分化为原始生殖细胞介导生产转基因鸡方法。具体做法如下：
前期通过OCT4,SOX2,NANOG,LIN28A慢病毒过表达载体将鸡成纤维细胞诱导重编程为iPS，然后通过BMP4/BMP8b/EGF体系将鸡iPS细胞诱导分化为PGC-like细胞，然后其注射至孵化2.5天的受体鸡胚血管，封口后继续孵化至出壳（记为F0代）， F0代嵌合体鸡饲养至性成熟，公母鸡进行交配产生F1代，观察并记录F1代出壳时羽色及胫色等表型性状，统计阳性后代鸡比例，并对其进行翅静脉采血，提取血液基因组进行基因组测序或微卫星位点检测。结果发现能够F1代鸡中存在阳性转基因鸡。
本发明切实可靠，严谨准确，成效显著，为研究分析体外转分化形成的PGC-like细胞产生转基因鸡的能力提供了一种新颖可行且高效的实验方法。
3、有益效果
本发明严谨准确，切实可行，应用广泛。 PGCs由于其生殖细胞的发育潜能，能够作为种质资源进行保护和利用，也可以作为基因编辑的目的细胞生产基因编辑动物，通过禽类体细胞转分化形成的PGC-like细胞异体/异种间移植产生后代。鸡是非哺乳动物中研究遗传学最重要的模式生物之一。因此，本发明的实验方法不仅在家禽上种质资源保护过程中可行，在脊椎动物的其他研究领域也可应用。这一发明将为体外诱导的PGC-like细胞介导后代鸡的生产研究提供更多的理论基础和技术支撑，也有利于对禽类种质资源进行保护和利用，同时也有利于转基因鸡的生产研究。
具体实施方式
本方法主要包括三个步骤：
1. OCT4/SOX2/NANOG/LIN28A四因子体系诱导鸡CEF重编程为iPS
2. BMP4/BMP8b/EGF诱导鸡iPS分化为iPGC
3. 鸡iPGC血管注射介导转基因鸡生产
具体操作如下：
1. OCT4/SOX2/NANOG/LIN28A四因子体系诱导鸡CEF重编程为iPS
(1)将生长状态良好的CEF接种于24孔板中，用含OCT4、SOX2、NANOG/、LIN28A慢病毒的无血清DMEM培养基侵染成纤维细胞。
(2)72小时后观察EGFP绿色荧光表达情况并将培养基更换为iPS诱导培养基。
(3)每两日更换一次诱导培养基，诱导至第21天时收集鸡iPS细胞进行荧光定量、免疫荧光、AKP染色等鉴定。
诱导鸡iPS分化为iPGC
(1)收集鸡iPS细胞，接种于100mm培养皿中，差速贴壁45分钟后收集上清，去除iPS中的饲养层细胞。
(2)将iPS细胞接种于孔板中，用BMP4/BMP8b/EGF诱导培养基进行培养。诱导第6天时收集鸡iPGC进行荧光定量、免疫荧光和PAS糖原染色等鉴定。
鸡iPGC血管注射介导转基因鸡生产
(1)取发育至2.5天的隐性白羽鸡种蛋，在钝端开口并将其放置在体式显微镜下，找到鸡胚血管。用显微注射针向血管中注射iPGC细胞悬液。
(2)将20μL青链霉素溶液从开口中轻轻滴入。用医用纸胶带将开口封住，将种蛋移回孵化箱中孵化至出壳（F0代）。
(3)F0代饲养至性成熟，公母鸡进行交配，收集种蛋（F1代）孵化。
(4)观察并记录F1代出壳时羽色及胫色等表型性状，统计阳性后代鸡比例。
(5)F1代鸡1月龄时对其进行翅静脉采血，提取血液基因组进行基因组测序或微卫星位点检测。
Claims (2)
1.一种鸡成纤维细胞转分化为原始生殖细胞介导生产转基因鸡的方法，其特征是，通过OCT4,SOX2,NANOG,LIN28A诱导鸡CEF重编程为iPS；通过BMP4/BMP8b/EGF诱导鸡iPS分化为iPGC；鸡iPGC血管注射介导转基因鸡生产。
2.根据权利要求1所述的一种鸡成纤维细胞转分化为原始生殖细胞介导生产转基因鸡的方法，其特征是是,具体做法为：
前期通过OCT4,SOX2,NANOG,LIN28A慢病毒过表达载体将鸡成纤维细胞诱导重编程为iPS；
然后通过BMP4/BMP8b/EGF体系将鸡iPS细胞诱导分化为PGC-like细胞；
然后其注射至孵化2.5天的受体鸡胚血管，封口后继续孵化至出壳，记为F0代，F0代嵌合体鸡饲养至性成熟，公母鸡进行交配产生F1代，观察并记录F1代出壳时羽色及胫色等表型性状，统计阳性后代鸡比例，并对其进行翅静脉采血，提取血液基因组进行基因组测序或微卫星位点检测，结果发现能够F1代鸡中存在阳性转基因鸡。
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