利用CRISPR/Cas9技术构建ALK7~(LoxP/LoxP)小鼠品系
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摘要
目的利用CRISPR/Cas9技术将LoxP序列靶向导入小鼠活化素受体样激酶7(ALK7)基因,构建ALK7LoxP/LoxP小鼠,与组织特异性Cre小鼠杂交,获得时空特异性ALK7基因敲除小鼠,为研究ALK7在特定时间特定组织中的功能奠定基础。方法利用CRISPR/Cas9技术编辑小鼠ALK7基因:设计合成识别ALK7基因外显子4-6上下游非编码序列的sg RNA。设计并合成LoxP-ALK7-LoxP打靶载体,测序正确后,显微注射法将体外合成的sg RNA、Cas9 m RNA和打靶载体注射到小鼠受精卵,移植受精卵至假孕小鼠输卵管代孕。获得仔鼠后通过PCR、Southern blot鉴定子代小鼠基因型,实时荧光定量PCR、Western blot检测ALK7转录表达水平。结果获得了含有目的基因的打靶阳性小鼠,并且插入的LoxP序列不影响ALK7的转录表达水平。结论利用CRISPR/Cas9技术成功将LoxP序列靶向引入小鼠ALK7基因,成功构建ALK7LoxP/LoxP小鼠品系,为进一步构建组织特异性ALK7基因敲除小鼠模型奠定了基础。
Aim To generate mutant mouse with ALK7 gene inserted by LoxP sequences by CRISPR/Cas9,to further develop temperospatial specific ALK7 deleted mouse models via cross bred with the tissue-specific Cre mouse,providing a basis for functional study of ALK7 in special time and tissue.Methods The CRISPR/Cas9 technology was used to edit ALK7.Two sg RNAs were designed to direct Cas9 endonuclease cleavage in intron 3-4 and intron 6-7.The targeting vector with LoxP-ALK7-LoxP sequence was designed.sg RNA,Cas9 m RNA and targeting vector were co-injected into zygotes,which were transferred to pseudopregnant mice.The pups were genotyped by PCR and Southern blott.Real-time PCR and Western blot were performed for analysis of the expression of ALK7.Results The ALK7LoxP/LoxP mouse was generated by CRISPR/Cas9,and did not show influence on the expression of ALK7.Conclusion The CRISPR/Cas9 technology can successfully generate the ALK7LoxP/LoxPmouse by inserting LoxP sequence into ALK7 gene,which is a basis for the creation of tissue-specific ALK7 deleted mouse models.
Aim To generate mutant mouse with ALK7 gene inserted by LoxP sequences by CRISPR/Cas9,to further develop temperospatial specific ALK7 deleted mouse models via cross bred with the tissue-specific Cre mouse,providing a basis for functional study of ALK7 in special time and tissue.Methods The CRISPR/Cas9 technology was used to edit ALK7.Two sg RNAs were designed to direct Cas9 endonuclease cleavage in intron 3-4 and intron 6-7.The targeting vector with LoxP-ALK7-LoxP sequence was designed.sg RNA,Cas9 m RNA and targeting vector were co-injected into zygotes,which were transferred to pseudopregnant mice.The pups were genotyped by PCR and Southern blott.Real-time PCR and Western blot were performed for analysis of the expression of ALK7.Results The ALK7LoxP/LoxP mouse was generated by CRISPR/Cas9,and did not show influence on the expression of ALK7.Conclusion The CRISPR/Cas9 technology can successfully generate the ALK7LoxP/LoxPmouse by inserting LoxP sequence into ALK7 gene,which is a basis for the creation of tissue-specific ALK7 deleted mouse models.
引文
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[14]Balkow A,Jagow J,Haas B,et al.A novel crosstalk between ALK7 and c GMP signaling differentially regulates brown adipocyte function[J].Mol Metab,2015,4(8):576-583.
[15]Mali P,Esvelt KM,Church GM.Cas9 as a versatile tool for engineering biology[J].Nat Methods,2013,10(10):957-963.
[16]Jinek M,Chylinski K,Fonfara I,et al.A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[J].Science,2013,337(6096):816-821.
[17]Zhao F,Huang F,Tang M,et al.Nodal induces apoptosis through activation of the ALK7 signaling pathway in pancreatic INS-1 beta-cells[J].Am J Physiol Endocrinol Metab,2012,303(1):E132-143.
[18]Yadav H,Quijano C,Kamaraju AK,et al.Protection from obesity and diabetes by blockade of TGF-beta/Smad3signaling[J].Cell Metab,2011,14(1):67-79.
[19]Burgess DJ.Technology:a CRISPR genome-editing tool[J].Nat Rev Genet,2013,14(2):80.
[2]Chiasson JL,Le Lorier J.Glycaemic control,cardiovascular disease,and mortality in type 2 diabetes[J].Lancet,2014,384(9958):1906-1907.
[3]Henninger AM,Eliasson B,Jenndahl LE,et al.Adipocyte hypertrophy,inflammation and fibrosis characterize subcutaneous adipose tissue of healthy,non-obese subjects predisposed to type 2 diabetes[J].PLoS One,2014,9(8):e105262.
[4]王芝嫣,王毅.脂肪组织异常与动脉粥样硬化[J].国际心血管病杂志,2017,44(05):271-273,281.
[5]Ryden M,Imamura T,Jornvall H,et al.A novel type I receptor serine-threonine kinase predominantly expressed in the adult central nervous system[J].J Biol Chem,1996,271(48):30603-30609.
[6]Liu L,Ding WY,Zhao J,et al.Activin receptor-like kinase7 mediates high glucose-induced H9c2 cardiomyoblast apoptosis through activation of Smad2/3[J].Int J Biochem Cell Biol,2013,45(9):2027-2035.
[7]Hu T,Su F,Jiang W,et al.Overexpression of activin receptor-like kinase 7 in breast cancer cells is associated with decreased cell growth and adhesion[J].Anticancer Res,2017,37(7):3441-3451.
[8]Khalil AM,Dotimas H,Kahn J,et al.Differential binding activity of tgf-beta family proteins to select tgf-beta receptors[J].J Pharmacol Exp Ther,2016,358(3):423-430.
[9]汪雄,张玲,陶凌.GDF11通过上调ABCA1表达促进小鼠体内胆固醇逆转运[J].中国动脉硬化杂志,2018,26(4):329-334.
[10]Zhang W,Wang H,Zhang W,et al.ALK7 gene polymorphism is associated with metabolic syndrome risk and cardiovascular remodeling[J].Arq Bras Cardiol,2013,101(2):134-140.
[11]Li WB,Zhao J,Liu L,et al.Silencing of activin receptorlike kinase 7 alleviates aortic stiffness in type 2 diabetic rats[J].Acta Diabetol,2015,52(4):717-726.
[12]Murakami M,Shirai M,Ooishi R,et al.Expression of activin receptor-like kinase 7 in adipose tissues[J].Biochem Genet,2013,51(3-4):202-210.
[13]Yogosawa S,Mizutani S,Ogawa Y,et al.Activin receptorlike kinase 7 suppresses lipolysis to accumulate fat in obesity through downregulation of peroxisome proliferatoractivated receptor gamma and C/EBP alpha[J].Diabetes,2013,62(1):115-123.
[14]Balkow A,Jagow J,Haas B,et al.A novel crosstalk between ALK7 and c GMP signaling differentially regulates brown adipocyte function[J].Mol Metab,2015,4(8):576-583.
[15]Mali P,Esvelt KM,Church GM.Cas9 as a versatile tool for engineering biology[J].Nat Methods,2013,10(10):957-963.
[16]Jinek M,Chylinski K,Fonfara I,et al.A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[J].Science,2013,337(6096):816-821.
[17]Zhao F,Huang F,Tang M,et al.Nodal induces apoptosis through activation of the ALK7 signaling pathway in pancreatic INS-1 beta-cells[J].Am J Physiol Endocrinol Metab,2012,303(1):E132-143.
[18]Yadav H,Quijano C,Kamaraju AK,et al.Protection from obesity and diabetes by blockade of TGF-beta/Smad3signaling[J].Cell Metab,2011,14(1):67-79.
[19]Burgess DJ.Technology:a CRISPR genome-editing tool[J].Nat Rev Genet,2013,14(2):80.
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