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| Construction of TPH1 Knockout Goat (Capra hircus) Mammary Epithelial Cell Lines by CRISPR/CAS9 System |
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Abstract Abstract 5-hydroxytryptamine (5-HT) is essential to stimulate skeletal calcium reabsorption for milk synthesis by the mammary gland. Tryptophan hydroxylase (TPH1) is the rate-limiting enzyme in peripheral 5-hydroxytryphtamine (5-HT) synthesis and thus plays an essential role in 5-HT metabolism. Objective of this study is to generate stable TPH1 gene knockout goat (Capra hircus) mammary epithelial cell line by CRISPR/Cas9 system, which could be an important material for exploring functions of 5-HT and calcium circulatory metabolism in goat mammary glands. Firstly, single guide RNA (sgRNA) sequences were designed based on the sequence of TPH1 (GenBank No.: 102184739) and were inserted into pX458 and pX459 vectors, respectively. Then goat mammary epithelial cells (GMECs) were transfected with pCas-guide vectors and puromycin (1 μg/mL) was used for screening positive cells. Finally, Western blot and DNA sequencing were used for identifying if the TPH1 gene of the GMECs had been knocked out. In this study, 3 pairs of pCAS-sgRNA vectors targeting TPH1 gene exon 1 of dairy goat were designed and constructed by using CRISPR/CAS9 technique. The transfection efficiency reached 20%; Single cell line px459-sg2-SC5 was screened by puromycin and the editing efficiency was 32%; DNA sequencing result showed that there were 10 bp base deletion at sg2 targeting site. Protein expression of TPH1 was blocked. Taken together, goat mammary epithelial cell line with stable knockout of TPH1 gene was successfully obtained, which would provide important materials for the study of the regulation of mammary gland physiology by 5-HT.
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Received: 11 December 2017
Published: 20 July 2018
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