农业生物技术学报2017，Vol.25Issue(8)：1366-1373,8.DOI:10.3969/j.issn.1674-7968.2017.08.017
粤黄鸡PGCs无饲养层培养体系的建立
Establishment of Feeder-free Culture System for PGCs of Yuehuang Chicken (Gallus gallus)
摘要
Abstract
Primordial germ cells (PGCs) are precursor cells of gonocyte,as a kind of pluripotent stem cell,PGCs have a similar morphology and multilineage potential with the embryonic stem (ES) cell.It can be cultured and subcultered in vitro and maintain its undifferentiated state and pluripotency under certain conditions.At present,PGCs are often cultured with feeder cells and serum,which causes difficulties and confusion for the later research.In order to establish a feeder-free culture system for PGCs in poultry,PGCs were isolated and cultured in vitro by improving the culture system in this study.The results showed that the PGCs with no feeder layer were 6～8 μm in diameter and the growth curve showed "S" shape.The population doubling time was (3.75±0.15) d.In the course of in vitro culture,PGCs could have better self-renewal and proliferation in the absence of feeder layer system conditions.PGCs could be cultured in vitro and maintain their undifferentiated state.Compared with similar studies this study not only maintained PGCs differentiation potential,but also obtained a large number of cells.The absence of feeder layer system could make PGCs maintain good growth status and had strong cell viability.The specific genes stage-specific embryonic antigen1 (SSEA-1) and deleted in azoospermia-like (DAZL) of PGCs were identified by immunofluorescence,and the results showed that both of them were expressed in PGCs,the cells stained by chemical immunization could observe the green fluorescence.The purified PGCs were labeled with PKH26 and cultured by feeder-free layer,then they were injected into the dorsal aorta of recipient chicken embryo that was hatched to the 17～20 stage until 5.5 d.The results showed that the PGCs which were isolated from chicken embryo had the same characteristics of itself after PKH26 labeling,which could be colonized in chicken embryo gonads with the blood circulation of chicken embryo.PGCs in vitro culture system had been optimized to ensure the undifferentiated state of it,since in vitro culture of PGCs was easier to differentiate.At present,the cultivation of PGCs was mainly based on Dulbecco's Modified Eagle Medium (DMEM) or Tumor Conditioned Medium (TCM),while a certain proportion of fetal bovine serum,amino acids and cells growth factors had been added on the basis of the feeder cells.But both the demanding preparation and a variety of cytokines or other components of the feeder cells had turned into a great obstacle for the research on PGCs proliferation,migration and other molecular mechanisms.This study optimized culture conditions of PGCs in vitro and broke the traditional culture method with serum and feeder layer and established a serum-free culture system without feeder layer.This culture system could avoid the adverse effects of the heterologous cells as feeder layer and the damage to cells from the drug residues of feeder layer.The composition of the culture medium was clear and it was convenient to study the molecular mechanism of cell differentiation and provides important technical support for application of PGCs in PGCs-based modem preservation technology,transgenic technology and developmental biology of poultry.关键词
原始生殖细胞(PGCs)/无饲养层/PKH26/胚胎注射Key words
Primordial germ cells (PGCs)/Feeder-free/PKH26/Embryo injection分类
农业科技引用本文复制引用
张晓芳,赵姝灿,张豫,郑桂纯,李京华,王丙云,陈志胜..粤黄鸡PGCs无饲养层培养体系的建立[J].农业生物技术学报,2017,25(8):1366-1373,8.基金项目
国家自然科学基金(No.31201871) （No.31201871）
