牦牛六个多能性相关转录因子OSKMNL的克隆和多顺反子慢病毒载体的构建

doi:10.11843/j.issn.0366-6964.2024.04.021

Abstract

Abstract: The purpose of this study was to clone 6 pluripotency-related transcription factors Oct4, Sox2, Klf4, c-Myc, Nanog, Lin28 (OSKMNL) of yak and construct polycistronic lentiviral vectors FUW-teto-OSM-EGFP and FUW-teto-KNL-mCherry. In this study, the reproductive crest tissue of a 3-5-month-old healthy female yak fetus was used as the research material. The complete coding region sequences of 6 pluripotent related transcription factors OSKMNL were cloned by RT-PCR and analyzed by bioinformatics. Lentiviral vectors FUW-teto-OSM-EGFP and FUW-teto-KNL-mCherry were constructed by seamless cloning. The lentivirus was packaged with 293T cells, and the packaged lentivirus was infected with 293T cells and yak fibroblasts. The virus infection was detected by fluorescence expression and RT-PCR technique (the experiment was divided into virus infection group and blank control group, each group had 3 repeats). The results showed that the coding region of cloned yak OSKMNL gene was 1 083, 963, 1 434, 1 320, 903 and 618 bp, respectively. Sequence analysis showed that the amino acid sequence of OSKMNL in yak shared more than 99% homology with cattle, and the homology of Sox2, Klf4, c-Myc with cattle was 100%. The results of evolutionary tree showed that yak had the closest genetic relationship with cattle, zebu cattle and buffalo, but the farthest relationship with mice. Protein structure prediction showed that the 6 transcription factors OSKMNL of yak all had the corresponding protein functional structures of the gene family, such as POU domain, HOX domain, HMG domain, Znf-C2H2 domain, HLH domain, CSP domain and so on. Lentiviral vectors FUW-teto-OSM-EGFP and FUW-teto-KNL-mCherry were constructed. 293T cells infected by lentivirus were found to express red and green fluorescence. Yak fibroblasts infected with lentivirus showed red and green fluorescence in the experimental group, and RT-PCR results showed bands with expected size. In this study, 6 pluripotency related transcription factors OSKMNL of yak were successfully cloned. Lentiviral vectors FUW-teto-OSM-EGFP and FUW-teto-KNL-mCherry were constructed, which contain 3 transcription factors of yak, respectively. Lentivirus could infect yak fibroblasts. It is helpful to promote the application of pluripotent related transcription factor OSKMNL in yak stem cells and prepare for the follow-up study of yak iPSC.

Key words: yak, gene cloning, sequence analysis, lentiviral vector

CLC Number:

S823.85

HUANG Xianpeng, XING Jiayi, BAI Yuanyuan, JIANG Yuting, MA Zhiwei, FU Wei, LAN Daoliang. Cloning of Six Pluripotent Related Transcription Factors OSKMNL in Yak and Construction of Polycistron Lentiviral Vector[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(4): 1579-1591.

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Cloning of Six Pluripotent Related Transcription Factors OSKMNL in Yak and Construction of Polycistron Lentiviral Vector

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