title: Establishment and characterization of buffalo fetal fibroblasts induced with human telomerase reverse transcriptase.
abstract: Fetal fibroblasts are often used as donor cells for SCNT, but their short lifespan greatly limits this application. To provide stable and long-lifespan cells, buffalo fetal fibroblasts (BFFs) transfected with human telomerase reverse transcriptase (hTERT). The hTERT-transfected BFFs (hTERT-BFFs) were evaluated by qRT-PCR, Western blot, karyotype analysis, telomerase activity assay, growth curve assay, flow cytometry, and soft agar assay. The development of SCNT embryos derived from hTERT-BFFs was also assessed in vitro. The morphology of hTERT-BFFs was similar to the nontransfected BFFs, and the karyotype of hTERT-BFFs was normal at passage 30. The hTERT-BFFs at passage 4 and 30 had higher telomerase activity and extended proliferative lifespan with an increase in cell population at S phase when compared with nontransfected BFFs at passage 5 and 30. The mRNA expression of p53 in hTERT-BFFs at passage 5 and 30 remained unchanged when compared with nontransfected BFFs at passage 5, whereas the mRNA expression of p53 in the nontransfected BFFs at passage 30 was increased. Soft agar assay showed that hTERT-BFFs at passage 30 were not a malignant phenotype. Significantly, more SCNT embryos derived from hTERT-BFFs at passage 5 and 30 developed to blastocysts in comparison with BFFs at passage 30. The Caudal type homeobox 2 and Connexin 43 genes were indicated to involve in the development of cloned embryos. These results indicate that transfection of BFFs with hTERT can extend their lifespan and retain their basic and key biological characteristics in the status of primary BFFs.
authors.affiliation: Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, China; Department of Reproductive Medical Center, The Affiliated Hospital of Guilin Medical University, Guilin, China.
authors.forename: Shun
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Department of Reproductive Medical Center, People's Hospital of Luohu District, Shenzhen, China.
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Guan
authors.affiliation: Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, China.
authors.forename: Fenghua
authors.initials: F
authors.lastname: Lu
authors.affiliation: Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, China.
authors.forename: Jianrong
authors.initials: J
authors.lastname: Jiang
authors.affiliation: Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, China.
authors.forename: Yanfei
authors.initials: Y
authors.lastname: Deng
authors.affiliation: Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, China.
authors.forename: Chan
authors.initials: C
authors.lastname: Luo
authors.affiliation: Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, China. Electronic address: ardsshi@gxu.edu.cn.
authors.forename: Deshun
authors.initials: D
authors.lastname: Shi
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000196:embryology; D001755:Blastocyst / Q000502:physiology; D002020:Buffaloes / Q000196:embryology*; D019976:Cloning, Organism / Q000379:methods; D047108:Embryonic Development; D005333:Fetus / Q000166:cytology; D005347:Fibroblasts / Q000201:enzymology*; D015870:Gene Expression; D006801:Humans; D053652:Nuclear Transfer Techniques; D012333:RNA, Messenger / Q000032:analysis; D019098:Telomerase / Q000235:genetics*; D014162:Transfection / Q000662:veterinary*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
chemical_list: D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D019098:Telomerase
keywords: Buffalo fetal fibroblast; Donor cell; Lifespan; Somatic cell nuclear transfer; hTERT