利用CRISPR/CAS9技术构建山羊乳腺上皮细胞TPH1基因敲除细胞系
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摘要
五羟色胺(5-hydroxytryphtamine,5-HT)是机体重要的单胺类神经递质,广泛分布于动物体各组织器官并参与机体多种生理功能。色氨酸羟化酶(tryptophan hydroxylase,TPH)1是5-HT合成的限速酶,在5-HT调控机体代谢过程中起关键作用。本研究旨在利用CRISPR/CAS9技术建立稳定敲除TPH1基因的山羊(Capra hircus)乳腺上皮细胞系,为研究山羊乳腺中5-HT调控山羊乳腺上皮细胞(goat mammary epithelial cells,GMEC)钙离子代谢的分子机理提供实验材料。首先根据Gen Bank中山羊TPH1基因序列(Gen Bank No.:102184739),设计靶向TPH1基因第一外显子的单导链RNA(single guide RNA,sgRNA),用pX458和pX459分别构建重组真核表达载体pX458-sgRNA和pX459-sgRNA,将重组质粒转染山羊乳腺上皮细胞,使用嘌呤霉素(1μg/mL)进行阳性细胞筛选,挑取单克隆细胞进行培养,用Western blot、T7E1酶切、基因组DNA测序等方法鉴定基因敲除效果。结果显示:通过CRISPR/CAS9技术成功在TPH1基因第一外显子打靶产生10 bp碱基缺失。本研究成功构建并筛选获得了TPH1基因稳定敲除的单克隆山羊乳腺上皮细胞系,为5-HT调控乳腺生理的功能研究提供了重要材料。
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(Gen Bank No.:102184739) and were inserted into pX458 and pX459 vectors,respectively.Then goat mammary epithelial cells(GMECs) were transfected with p Cas-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 p CASsgRNA 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.
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(Gen Bank No.:102184739) and were inserted into pX458 and pX459 vectors,respectively.Then goat mammary epithelial cells(GMECs) were transfected with p Cas-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 p CASsgRNA 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.
引文
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Li J,Shou J,Guo Y,et al.2015.Efficient inversions and duplications of mammalian regulatory DNA elements and gene clusters by CRISPR/Cas9[J].Journal of Molecular Cell Biology,7(4):284-298.
Lillest?l R K,Redder P,Garrett R A,et al.2006.A putative viral defence mechanism in archaeal cells[J].Archaea,2(1):59-72.
Makarova K S,Haft D H,Barrangou R,et al.2011a.Evolution and classification of the CRISPR-Cas systems[J].Nature Reviews,9(6):467-477.
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O'Geen H,Henry I M,Bhakta M S,et al.2015.A genomewide analysis of Cas9 binding specificity using Ch IP-seq and targeted sequence capture[J].Nucleic Acids Research,43(6):3389-3404.
Pai V,Horseman N D,et al.2008.Biphasic regulation of mammary epithelial resistance by 5-HT through activation of multiple pathways[J].Journal of Biologica Chemistry,283(5):30901-30910.
Pattanayak V,Lin S,Guilinger J P,et al.2013.High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity[J].Nature Biotechnology,31(9):839-843.
Raymond J R,Mukhin J V,Gelasco A,et al.2001.Multiplicity of mechanisms of serotonin receptor signal transduction[J].Pharmacology Therapeutics,92(2):179-212.
Shalem O,Sanjana N E,Hartenian E,et al.2014.Genomescale CRISPR-Cas9 knockout screening in human cells[J].Science,343(6166):84-87.
Thomas D R,Hagan J J,et al.2004.5-HT7 receptors[J].Current Drug Targets CNS Neurological&Disorders,3(1):81-90.
Trevino A E,Zhang F,et al.2014.Genome editing using Cas9nickases[J].Methods in Enzymology,546(546C):161-174.
Wang Y,Cheng X,Shan Q,et al.2014.Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew[J].Nature Biotechnol,32(9):947-951.
Wu X,Kriz A J,Sharp P A,et al.2014.Target specificity of the CRISPR-Cas9 system[J].Quantitative Biology,2(2):59-70.
Sari Y,Zhou F C,et al.2003.Serotonin and its transporter on proliferation of fetal heart cells[J].International Journal of Developmental Neuroscience,21(8):417-424.
Zang W J,Li H,Zhang Z F,et al.2018.Serotonin induces parathyroid hormone-related protein in goat mammary gland[J].Journal of Animal Science,96(3):1010-1016.
Barrangou R,Fremaux C,Deveau H,et al.2007.CRISPR provides acquired resistance against viruses in prokaryotes[J].Science,315(5819):1709-1712.
Coffey A,Ross R P.2002.Bacteriophage-resistance systems in dairy starter strains,molecular analysis to application[J].Anton Leeuw,82(1):303-321.
Deltcheva E,Chylinski K,Sharma C,et al.2011.CRISPRRNA maturation by trans-encoded small RNA and hos factor RNase III[J].Nature,471(7340):602-607.
Pierre D W,Pespeni M H,Palumbi S R,et al.2015.SNP genotyping and population genomics from expressed sequences-current advances and future possibilities[J]Molecular Ecology,3(21):13165.
Ran F A,Hsu P D,Zhang F et al.2013.Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity[J].Cell,154:1380-1389.
Garneau J E,Dupuis M E,Villion M,et al.2010.The CRIS-PR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA[J].Nature,468(7320):67-71.
Gesner E M,Schellenberg M J,Garside E L,et al.2011.Recognition and maturation of effector RNAs in a CRISPRinterference pathway[J].Nature Structural&Molecular Biology,18(6):688-692
Helen S.2014.CRISPR technology leaps from lab to industry[J].Journal of Nematology,46(2):130-260.
Hernandez L L,Stiening C M,Wheelock J B,et al.2008Evaluation of serotonin as a feedback inhibitor of lactation in the bovine[J].Journal of Dairy Science,91(5)1834-1844.
Hernandez L L,Limes S W,et al.2009.The bovine mammary gland expresses multiple functional isoforms of serotonin receptors[J].Journal of Endocrinology,203(1):123-131.
Ishino Y,Shinagawa H,Makino K et al.1987.Nucleotide sequence of the iap gene,responsible for alkaline phosphatase isozyme conversion in Escherichia coli and identification of the gene product[J].Journal of Bacteriology,169(12):5429-5433.
Jansen R,Van J E,Gaastra W,et al.2002.Identification of genes that are associated with DNA repeats in prokaryotes[J].Molecular microbiology,43(6):1565-1575.
Laporta J,Keil K P,Vezina C M,Hernandez L L,et al.2014.Peripheral serotonin regulates maternal calcium trafficking in mammary epithelial cells during lactation in mice[J].Plo S One;9:e110190.
Laporta J,Moore S A,Weaver S R,et al.2015.Increasing serotonin concentrations alter calcium and energy metabolism in dairy cows[J].Journal of Endocrinology,226(1):43-55.
Li J,Shou J,Guo Y,et al.2015.Efficient inversions and duplications of mammalian regulatory DNA elements and gene clusters by CRISPR/Cas9[J].Journal of Molecular Cell Biology,7(4):284-298.
Lillest?l R K,Redder P,Garrett R A,et al.2006.A putative viral defence mechanism in archaeal cells[J].Archaea,2(1):59-72.
Makarova K S,Haft D H,Barrangou R,et al.2011a.Evolution and classification of the CRISPR-Cas systems[J].Nature Reviews,9(6):467-477.
Makarova K S,Aravind L,Wolf Y I,et al.2011b.Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems[J].Biology Direct,6(38):1-27.
Marraffini L A,Sontheimer E J,Marraffini L A,et al.2008.CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA[J].Science,322(5909):1843-1845.
Rapport M M,Green A A,Page I H,et al.1948.Serum vasoconstrictor(serotonin).IV.Isolation and characterization[J].The Journal of Biological Chemistry,176(3):1243-1251.
Moyer T C,Holland A J,et al.2015.Generation of a conditional analog-sensitive kinase in human cells using CRISPR/Cas9-mediated genome engineering[J].Methods in Cell Biology,129:19-36.
Nishimasu H,Ran F A,Hsu P D,et al.2014.Crystal structure of Cas9 in complex with guide RNA and target DNA[J]Cell,156(5):935-949.
O'Geen H,Henry I M,Bhakta M S,et al.2015.A genomewide analysis of Cas9 binding specificity using Ch IP-seq and targeted sequence capture[J].Nucleic Acids Research,43(6):3389-3404.
Pai V,Horseman N D,et al.2008.Biphasic regulation of mammary epithelial resistance by 5-HT through activation of multiple pathways[J].Journal of Biologica Chemistry,283(5):30901-30910.
Pattanayak V,Lin S,Guilinger J P,et al.2013.High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity[J].Nature Biotechnology,31(9):839-843.
Raymond J R,Mukhin J V,Gelasco A,et al.2001.Multiplicity of mechanisms of serotonin receptor signal transduction[J].Pharmacology Therapeutics,92(2):179-212.
Shalem O,Sanjana N E,Hartenian E,et al.2014.Genomescale CRISPR-Cas9 knockout screening in human cells[J].Science,343(6166):84-87.
Thomas D R,Hagan J J,et al.2004.5-HT7 receptors[J].Current Drug Targets CNS Neurological&Disorders,3(1):81-90.
Trevino A E,Zhang F,et al.2014.Genome editing using Cas9nickases[J].Methods in Enzymology,546(546C):161-174.
Wang Y,Cheng X,Shan Q,et al.2014.Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew[J].Nature Biotechnol,32(9):947-951.
Wu X,Kriz A J,Sharp P A,et al.2014.Target specificity of the CRISPR-Cas9 system[J].Quantitative Biology,2(2):59-70.
Sari Y,Zhou F C,et al.2003.Serotonin and its transporter on proliferation of fetal heart cells[J].International Journal of Developmental Neuroscience,21(8):417-424.
Zang W J,Li H,Zhang Z F,et al.2018.Serotonin induces parathyroid hormone-related protein in goat mammary gland[J].Journal of Animal Science,96(3):1010-1016.