tPA/gGH双基因转染山羊乳腺上皮细胞表达分析

doi:10.16288/j.yczz.19-299

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Hereditas(Beijing) ›› 2020, Vol. 42 ›› Issue (4): 380-387.doi: 10.16288/j.yczz.19-299

• Research Article • Previous Articles Next Articles

Expression analysis of tPA/gGH double genes in transfected goat mammary epithelial cells

Shaozheng Song¹, Kangying Yu¹, Ting Zhang², Rui Lu², Shengqiang Pan¹, Mingming Zhou¹(), Yong Cheng²()

^1. School of Nursing, Wuxi Taihu University, Wuxi 214000, China
^2. Jiangsu Provincial Research Center for Animal Transgenesis and Biopharming, College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China

Received:2019-09-24 Revised:2019-11-29 Online:2020-04-20 Published:2020-02-27
Contact: Zhou Mingming,Cheng Yong E-mail:zmm19770@126.com;chengyong12@yeah.net
Supported by:
Supported by the Natural Science Foundation of Colleges and Universities in Jiangsu Province Nos(17KJD310004);Supported by the Natural Science Foundation of Colleges and Universities in Jiangsu Province Nos(19KJB180030);National Major Special Projects on New Cultivation for Transgenic Organisms No(2014ZX08008-004)

Abstract

Abstract:

tPA is a thrombolytic agent widely used in clinical settings. While double gene co-integration into organisms can produce synergistic effects and improved expression levels of the target gene, there are few reports detailing the co-integration of the tPA and gGH genes and an increased expression level of tPA. In order to study this, we obtained monoclonal goat mammary epithelial cell lines with tPA/gGH double gene integration and we analyzed the tPA expression level of single and double gene integration cells. We constructed a mammary gland-specific expression vector PCL25/gGH by using the β-casein gene as the regulatory sequence. The tPA and gGH genes were co-transfected into goat mammary epithelial cells by electrotransfection. Resistant cell lines were screened by G418, and transgenic monoclonal cell lines were obtained by PCR detection. tPA expression was induced by prolactin and subsequently, the cell induction solution was assayed after 48 hours by ELISA and Western blotting. The results show that a total of 142 resistant monoclonal cells were obtained including 53 tPA monogenic integration cell lines and 34 tPA/gGH double gene integration cell lines. The rate of double gene integration was 23.9% (34/142). A total of 29 cells were detected to be able to express tPA, of which 12 were single-gene-expressing cells and the corresponding expression rate was 22.6% (12/53). There were 17 double-gene- expressing cells with a corresponding expression rate of 50.0% (17/34). The expression level of tPA in single-gene cells was 7.5-52.0 μg/mL, while in double-gene cells was 40-360 μg/mL, which was significantly greater than that in single- gene cells. The goat mammary epithelial cell lines with tPA/gGH gene integration were successfully obtained by electrotransfection, and we proved that the expression level of tPA in the double gene integration cell lines with tPA/gGH gene integration was significantly increased. Our findings lay the foundation for the additional study of highly expressed transgenic goats and other animals with determination of scientific and clinical utility.

Key words: tPA, double genes, transgene, goat mammary epithelial cells, expression

Shaozheng Song, Kangying Yu, Ting Zhang, Rui Lu, Shengqiang Pan, Mingming Zhou, Yong Cheng. Expression analysis of tPA/gGH double genes in transfected goat mammary epithelial cells[J]. Hereditas(Beijing), 2020, 42(4): 380-387.

Figures/Tables 7

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引物名称	引物序列(5°→3°)
gGH-F	CCGCTCGAGCGGATGATGGCTGCAGGCCCCCGGA
gGH-R	CCGCTCGAGCGGCTAGAAGGCACAGCTGGCCTCCCCG
CMV/tPA-F	CGTGGATAGCGGTTTGA
CMV/tPA-R	GAGCCCTCCTTTGATGC

细胞(142个)	整合细胞数	整合率(%)	表达细胞数	表达率(%)	表达水平(μg/mL)
tPA 单基因整合	53	37.3	12	22.6	7.5~52.0
tPA/gGH 双基因整合	34	23.9	17	50.0	40~360

Expression analysis of tPA/gGH double genes in transfected goat mammary epithelial cells

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