Received: 21 January 2018 | Accepted: 7 May 2018 DOI: 10.1002/jcb.27137 RESEARCH ARTICLE The establishment of clonally derived chicken embryonic fibroblast cell line (CSC) with high transfection efficiency and ability as a feeder cell Ruifeng Zhao 1 | Jing Jin 1 | Xinyu Sun 1 | Kai Jin 1 | Man Wang 1 | Mahmoud F. Ahmed 2 | Qisheng Zuo 1,3,4 | Yani Zhang 1,3,4 | Zhenhua Zhao 5 | Guohong Chen 1,3,4 | Bichun Li 1,3,4 1 Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, China 2 College of Veterinary Medicine, Suez Canal University, Ismailia, Egypt 3 Institutes of Agricultural Science and Technology Development, College of Animal Science and Technology, Yangzhou University, Yangzhou, China 4 Joint International Research Laboratory of Agriculture and Agri‐Product Safety of Ministry of Education of China, Yangzhou University, Yangzhou, China 5 Poultry Institute, Chinese Academy of Agricultural Sciences, Yangzhou, China Correspondence Bichun Li, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China. Email: yubcli@yzu.edu.cn Funding information National Natural Science Foundation of China, Grant/Award Numbers: 31472087, 31572390; College Students’ Innovation and Entrepreneurship Training Program, Grant/Award Number: Yangzhou University No. X20170702; The Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions for Funding the Study; Key Research and Development Program, Grant/Award Number: 2017YFE0108000; High Level Talents Support Program of Yangzhou University Abstract This study established a single cloned chicken embryonic fibroblast (CEF) cell line. It solves the main problem of the instability of a cultured primary cell and its impact on the experiment. In this study, CEF pass through this crisis and formed a continuous cell line after subculture. We isolated single postcrisis CEF by a mouth pipette under a convert microscope then established a single cloned cell line named CSC‐1‐5 which passaged continuously from 96‐well plates to 60 mm culture plates. CSC has a normal chicken diploid karyotype, no tumorigenicity, and a high G2/M phase cell ratio. We found that Fugene could mediate the transfection of CSCs efficiently; it was significantly improved compared with the primary cells. It could also promote the proliferation of chicken embryonic stem cell as a feeder layer. KEYWORDS chicken embryonic fibroblast, feed layer cells, mouth pipette, single clone, transfection 1 | INTRODUCTION Fibroblast is a type of cell differentiated from a mesench- ymal stem cell, which synthesizes the extracellular matrix and collagen. 1 It is important for animal tissue metabolism during embryo development. This kind of cell is often used for transfection or drug tests as target cells. 2,3 It is also used as feeder layer cells to promote the proliferation of stem cells after the inhibition of mitomycin C or Co60 rays. 4 Primary fibroblasts separated from tissue have many unique benefits for use as feeder cells, but they may exert several adverse effects on other experiments because of their low homology and unstable proliferation. Many continuous cell lines, such as 3T3, STO, DF‐1, etc have been established for stable proliferation. 3,5,6 They still cannot avoid miscellaneous cells because of its polyclonal origin. To solve this problem, many researches use a single cell to establish cell lines; this is called a single cloned cell line. These kinds of cell lines are in extremely high homology. Many approaches have been used to prepare the single cloned cell line, such as limit diluting, 7 clone J Cell Biochem. 2018;1‐10. wileyonlinelibrary.com/journal/jcb © 2018 Wiley Periodicals, Inc. | 1