OALib Journal期刊
ISSN: 2333-9721
费用：99美元

投递稿件

查看量	下载量

相关文章
更多...

- 2019

mSpag9肾脏特异性基因敲除小鼠模型的构建与鉴定 Generation of mSpag9 conditional knockout mice and phenotype identification

宋远,张璐,阳诚,颜奇,陈朝威,朱凯,王惠明

Full-Text Cite this paper Add to My Lib

Abstract:

目的:为研究支架蛋白JLP在肾脏中的生理功能,建立小鼠精子相关抗原(mSpag9)肾脏特异性基因敲除小鼠模型,为研究该基因所发挥的重要生理功能提供材料和思路。方法:构建mSpag9基因重组载体,通过电转打靶转染胚胎干细胞(ES细胞),将筛选出的阳性ES细胞克隆,经显微注射至超排小鼠的囊胚腔后移植于受体小鼠,从子代筛选嵌合体小鼠,与携带flp位点C57/BL6N小鼠杂交后得到mSpag9flox/+基因敲除小鼠,自交筛选出mSpag9flox/flox小鼠,再与KSP-CreERT2小鼠杂交得到mSpag9flox/flox-Cre肾脏特异性基因敲除小鼠并进行鉴定,待小鼠成长至5周,使用Tamoxifen(10 mg/mL葵花籽油溶解)腹腔注射(0.04 mg/g)诱导Cre重组酶特异性表达于肾小管上皮细胞。结果:经PCR扫描后得到10株阳性ES细胞克隆,其中6株使用Southern Blot鉴定;经显微注射后得到4只雄性、5只雌性嵌合体小鼠,与flp小鼠杂交后得到9只mSpag9flox/+基因敲除小鼠,将自交得到的mSpag9flox/flox小鼠与KSP-CreERT2小鼠交配筛选出mSpag9flox/+-Cre+3只小鼠,将其与mSpag9flox/flox小鼠回交得到基因型为mSpag9flox/flox-Cre+小鼠,即为mSpag9肾脏特异性基因敲除小鼠。结论:该模型利用Cre-Loxp系统实现基因mSpag9的基因打靶,Tamxifen诱导基因mSpag9实现该基因在肾小管上皮细胞上敲除,为进一步研究mSpag9基因及支架蛋白在肾脏生理病理进展中所发挥的作用提供工具

References

[1]	Kelkar N,Standen CL,Davis RJ.Role of the JIP4scaffold protein in the regulation of mitogen-activated protein kinase signaling pathways[J].Mol Cell Biol,2005,25(7):2 733-2 743.
[2]	Wang HM,Yan Q,Yang T,et al.Scaffold protein JLP is critical for CD40 signaling in B lymphocytes[J].J Biol Chem,2015,290(9):5 256-5 266.
[3]	付强,王惠明,颜奇,等.支架蛋白JLP在单侧输尿管梗阻小鼠肾间质纤维化中的作用及其机制探讨[J].中华肾脏病杂志,2016,32(1):30-36.Fu Q,Wang HM,Yan Q,et al.Role of scaffolding protein JLP on the progression of renal interstitial fibrosis in mice model of unilateral ureteral obstruction and its underlying mechanism[J].Chinese Journal of Nephrology,2016,32(1):30-36.
[4]	刘珊,王惠明,丁国华,等.支架蛋白JLP对人肾小管上皮细胞转分化的影响[J].中华肾脏病杂志,2016,32(8):612-616.Liu S,Wang HM,Yan Q,et al.Role of JLP on the epithelial to mesenchymal transition in renal tubular epithelial cells[J].Chinese Journal of Nephrology,2016,32(8):612-616.
[5]	Schmidt-Supprian M,Rajewsky K.Vagaries of conditional gene targeting[J].Nat Immunol,2007,8(7):665-668.
[6]	Dhanasekaran DN,Kashef K,Lee CM,et al.Scaffold proteins of MAP-kinase modules[J].Oncogene,2007,26(22):3 185-3 202.
[7]	Liu P,Jenkins NA,Copeland NG.A highly efficient recombineering-based method for generating conditional knockout mutations[J].Genome Res,2003,13(3):476-484.
[8]	Sedivy JM.Gene targeting comes to top-down drug screens[J].Trends Biotechnol,2002,20(3):92-93.
[9]	Pluck A,Klasen C.Generation of chimeras by microinjection[J].Methods Mol Biol,2009,561:199-217.
[10]	Shiina H,Matsumoto T,Sato T,et al.Premature ovarian failure in androgen receptor-deficient mice[J].Proc Natl Acad Sci U S A,2006,103(1):224-229.
[11]	Rojek A,Fuchtbauer EM,Kwon TH,et al.Severe urinary concentrating defect in renal collecting duct-selective AQP2 conditional-knockout mice[J].Proc Natl Acad Sci U S A,2006,103(15):6 037-6 042.
[12]	Lee CM,Onesime D,Reddy CD,et al.JLP:A scaffolding protein that tethers JNK/p38MAPK signaling modules and transcription factors[J].Proc Natl Acad Sci USA,2002,99(22):14 189-14 194.

Full-Text

Contact Us

service@oalib.com

QQ:3279437679

WhatsApp +8615387084133