Scientia Agricultura Sinica ›› 2014, Vol. 47 ›› Issue (2): 394-402.doi: 10.3864/j.issn.0578-1752.2014.02.019

• ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT • Previous Articles     Next Articles

Cloning of Buffalo (Bubalus bubalis) Oct4 and Nanog Genes and Ectopic Expression in Buffalo Fetal Fibroblasts

 DENG  Yan-Fei, LIU  Qing-You, DENG  Hai-Ying, LUO  Chan, YANG  Su-Fang, SHI  De-Shun   

  1. State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning 530005
  • Received:2013-06-09 Online:2014-01-15 Published:2013-09-26

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Abstract: 【Objective】Oct4 and Nanog were the pluripotency-related transcription factors, and play important roles in the differentiation of stem cell and embryo development. In this study, the buffalo Oct4 and Nanog genes were cloned, the gene sequences and protein sequences were analysed, and eukaryotic expression vectors were constructed. This study will provide a good foundation for investigating the functions of Oct4 and Nanog, especially their roles in the embryo development, establishing buffalo embryonic stem cell lines and inducing pluripotent stem cell lines (iPSC). 【Method】The RNA and DNA were extracted from fetus germ ridge and somatic cells, and the RNA was reverse-transcribed into first-strand cDNA. The primers were designed specifically according to the bovine gene sequences (Oct4：NM_174580，Nanog：NM_001025344). The coding sequences (CDS) and DNA fragments of Oct4 and Nanog were cloned by RT-PCR and PCR, respectively, three fragments for Oct4 gene and two fragments for Nanog. The protein structure prediction and homology comparison were analysed by online software. After digestion with enzymes (EcoRⅠ, XholⅠfor Oct4 and XholⅠ, NotⅠfor Nanog), the CDS of Oct4 and Nanog were inserted into the pMX retrovirus vector using T4 ligase, respectively, named pMX-Oct4 and pMX-Nanog. Buffalo fetal fibroblasts (BFFs) were cultured by the method of tissue explants. The retrovirus supernatant was collected from the retroviral system after virus packaging, and infected the BFFs 12-15 h. After culturing for 48 h, the ectopic expression of Oct4 and Nanog in the BFFs were detected by RT-PCR and immunofluorescence assay.【Result】The results showed that the CDS and DNA length of Oct4 was 1083 bp and 4509 bp, respectively, encoding 361 amino acids, and containing 5 exons and 4 introns. The CDS and DNA length of Nanog was 903 bp and 4473 bp, respectively, encoding 361 amino acids, and containing 4 exons and 3 introns. The gene sequences were submitted to GenBank online, and the accession numbers were JN991003 and JN991004, respectively. The amino acids of buffalo Oct4 and Nanog exhibited high homology with bovine, pig, human and mouse, the percentage of conservatism displayed Oct4 (98%, 96%, 91% and 81%) and Nanog (90%, 81%, 69% and 47%). Buffalo Oct4 and Nanog protein structures were similar to that of mice, Oct4 containing POU-domain and Nanog containing HOX-domain. Using the retrovirus vectors pMX-Oct4 and pMX-Nanog, buffalo Oct4 and Nanog genes could be transfected into BFFs, and their mRNA and protein could be expressed in the BFFs. 【Conclusion】 The CDS and DNA fragments of buffalo Oct4 and Nanog were cloned, their base sequence and amino acid sequences were conservation during evolution. Oct4 and Nanog were transfected into BFFs and expressed in BFFs using the retrovirus transgene method. The retrovirus-based target genes transfer could be used in the buffalo genetically modified research and buffalo iPSC generation.

Key words: buffalo , Oct4 gene , Nanog gene , cloning , ectopic expression

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