利用piggyBac转座子构建小鼠诱导性多能干细胞及其鉴定

doi:10.13481/j.1671-587x.20160408

吉林大学学报(医学版) ›› 2016, Vol. 42 ›› Issue (04): 676-680.doi: 10.13481/j.1671-587x.20160408

利用piggyBac转座子构建小鼠诱导性多能干细胞及其鉴定

任丽伟, 魏培, 杨晓菲, 杨璐, 邓春艳, 齐晖, 李富荣

暨南大学第二临床医学院广东省深圳市人民医院干细胞与细胞治疗重点实验室, 广东深圳 518020

收稿日期:2015-10-12 发布日期:2016-07-20
通讯作者: 李富荣,教授,博士研究生导师(Tel:0755-25533000-2450,E-mail:frli62@163.com) E-mail:frli62@163.com
作者简介:任丽伟(1989-),女,山东省聊城市人,医学硕士,主要从事干细胞和细胞治疗方面的研究。
基金资助:
国家自然科学基金资助课题（81270857）；广东省科技厅自然科学基金资助课题（S2013010014832）；广东省深圳市科技创新委员会科技计划项目资助课题（GJHZ20120618153934353，JCYJ2061853743791）

Construction of induced mouse pluripotent stem cells by piggyBac transposon and its identification

REN Liwei, WEI Pei, YANG Xiaofei, YANG Lu, DENG Chunyan, QI Hui, LI Furong

Key Laboratory of Stem Cell and Cellular Therapy, Shenzhen People's Hospital, Second Clinical Medical College, Jinan University, Shenzhen 518020, China

Received:2015-10-12 Published:2016-07-20

摘要/Abstract

摘要：

目的：利用 piggyBac 转座子载体携带鼠源 Oct4、Sox2、Klf4 和c-Myc 4 个核转录因子，重编程胎鼠成纤维细胞（MEFs）为诱导性多能干细胞（iPSCs），并探讨其作用效果。方法：分离Oct4-GFP小鼠的 MEFs，将携带鼠源 Oct4、Sox2、Klf4、c-Myc等4个基因的piggyBac 转座子转入至MEFs；观察iPSCs克隆形成过程的形态表现；分析iPSCs染色体组成，评价iPSCs的核型变化。RT-PCR法检测小鼠iPSCs中胚胎干细胞（ESCs）相关基因Oct4、Nanog和FGF4的表达；免疫荧光、碱性磷酸酶染色和流式细胞术检测小鼠iPSCs中SSEA-1和Nanog蛋白表达。将iPSCs接种到NOD-SCID小鼠腹股沟进行畸胎瘤实验，4周后取畸胎瘤制备组织切片进行HE染色，观察组织分化情况。结果：通过piggyBac转座子成功构建iPSCs，其具有正常的核型，形态与小鼠ESCs相似，克隆边界清晰、呈圆形或卵圆形，克隆内细胞致密、核大。RT-PCR 和免疫荧光染色分析，iPSCs可表达多能性基因和蛋白（Oct4、Sox2、Kif4和c-Myc）。在免疫缺陷小鼠体内接种iPSCs可形成具有3个胚层组织的畸胎瘤。结论：以piggyBac转座子为载体将Oct4、Sox2、Klf4 和c-Myc 4个转录因子基因导入MEFs，可以成功诱导MEFs成为具有ESCs特征的正常核型iPSCs。

关键词: piggyBac, 重编程, 小鼠胚胎纤维细胞, 诱导性多能干细胞

Abstract:

Objective: To investigate the effect of piggyBac transpon,as a carrier of four defined transcription factors Oct4,Sox2,Klf4 and c-Myc,in the reprogramming of mouse embryonic fibroblasts(MEFs) to induced pluripotent stem cells(iPSCs). Methods: The MEFs were isolated from Oct4-GFP fetal mice and transfected by piggyBac transposon with four factors(Oct4,Sox2,Klf4 and c-Myc). The morphological changes of clones were traced with microscope during the process of induction. The chromosomes were analyzed to evaluate the karyotypic variation of iPSCs. The mRNA expressions of Oct4,Nanog and FGF4 associated with embryonic stem cells(ESCs) in the iPSCs of mice were tested by RT-PCR;the protein expressions of SSEA-1,Nanog and Alkaline phosphatase in the iPSCs of mice were determined by flow cytometry,immunofluorescence and AP staining. The iPSCs were transplanted into the NOD-SCID mouse groin,4 weeks later,the teratomas were removed for HE staining and the differentiation of tissue was observed. Results: The iPSCs were successfully obtained from MEFs by piggyBac carrying Oct4,Sox2,Klf4, and c-Myc. The round or oval iPSCs clones were similar to ESCs with clear boundry and large dense nuleus. The iPSCs showed the normal karyotypic and expressed the marker genes (Oct4,Nanog and FGF4) and proteins (SSEA-1,Nanog and AP) of ESCs. Teratomas containing three germ layers were formed in NOD-SCID mice after tanspalantation of iPSCs. Conclusion: The iPSCs are reprogrammed from MEFs by piggyBac transposon with four transcription factors-Oct4,Sox2,Klf4 and c-Myc,and the iPSCs with normal karyotype possess the characteristics of ESCs.

Key words: piggyBac, reprogramming, mouse embryo fibroblasts, induced pluripotent stem cells

中图分类号:

Q813

任丽伟, 魏培, 杨晓菲, 杨璐, 邓春艳, 齐晖, 李富荣. 利用piggyBac转座子构建小鼠诱导性多能干细胞及其鉴定[J]. 吉林大学学报(医学版), 2016, 42(04): 676-680.

REN Liwei, WEI Pei, YANG Xiaofei, YANG Lu, DENG Chunyan, QI Hui, LI Furong. Construction of induced mouse pluripotent stem cells by piggyBac transposon and its identification[J]. Journal of Jilin University Medicine Edition, 2016, 42(04): 676-680.

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利用piggyBac转座子构建小鼠诱导性多能干细胞及其鉴定

Construction of induced mouse pluripotent stem cells by piggyBac transposon and its identification

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