X-盒结合蛋白1条件性基因敲除小鼠模型的建立
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摘要
目的建立可进行条件性敲除X-盒结合蛋白1(Xbp1)基因的flox杂合子小鼠。方法通过ET-clone的方法构建基于cre-loxp系统和FLP-frt系统的条件性基因打靶载体。载体线性化后电转JM8A3 ES细胞,经G418和Ganc筛选获得抗性ES细胞克隆。经长片段PCR鉴定获得正确同源重组的阳性克隆。阳性ES细胞经克隆扩增后,注射入C57BL/6J小鼠的囊胚中,获得嵌合鼠,筛选出高比例嵌合小鼠与野生型C57BL/6J小鼠交配后获得阳性F1代小鼠,并分别进行PCR和测序鉴定。结果成功构建打靶载体,共获得144个抗性ES细胞,克隆经长片段PCR鉴定,共获得4个正确同源重组的阳性克隆。获得4只阳性F1代小鼠,经测序鉴定正确。Xbp1基因flox杂合子小鼠表型无明显异常。结论成功构建了条件性敲除Xbp1基因的flox杂合子小鼠,为未来研究器官或组织特异性的Xbp1生物学作用奠定了基础。
Objective To establish the flox-heterozygous mouse model in which the X-box-binding protein 1( Xbp1) gene was conditionally knocked out. Methods The conditional gene-targeting vector,based on the cre-loxp system and the FLP-frt system,was constructed with the ET-clone method. The vectors were linearized and electroporated into the JM8A3 embryonic stem( ES) cells. The resistant ES cell clones were obtained through G418 and Ganc screening. The positive clones with correct homologousrecombination were obtained through identification with the long-range PCR method. Positive ES cells were clonally expanded,and injected into the blastocysts of C57 BL/6 J mice to obtain the chimeric mice. The high-rate chimeric mice were screened,and mated with the wild-type C57 BL/6 J mice to obtain the positive F1 mice,which were identified by PCR and sequencing. Results The targeting vectors were successfully constructed. A total of 144 resistant ES cell clones were obtained and identified by long-range PCR. Four positive homologous recombination clones were obtained. Four positive F1 mice were obtained and confirmed by sequencing. There was no obvious abnormality in the phenotypes of the Xbp1 gene flox-heterozygous mice. Conclusion The conditional knockout flox-heterozygous mouse model of Xbp1 gene was successfully established,which laid a foundation for further studies of the organ or tissue-specific biological effects of Xbp1 gene.
Objective To establish the flox-heterozygous mouse model in which the X-box-binding protein 1( Xbp1) gene was conditionally knocked out. Methods The conditional gene-targeting vector,based on the cre-loxp system and the FLP-frt system,was constructed with the ET-clone method. The vectors were linearized and electroporated into the JM8A3 embryonic stem( ES) cells. The resistant ES cell clones were obtained through G418 and Ganc screening. The positive clones with correct homologousrecombination were obtained through identification with the long-range PCR method. Positive ES cells were clonally expanded,and injected into the blastocysts of C57 BL/6 J mice to obtain the chimeric mice. The high-rate chimeric mice were screened,and mated with the wild-type C57 BL/6 J mice to obtain the positive F1 mice,which were identified by PCR and sequencing. Results The targeting vectors were successfully constructed. A total of 144 resistant ES cell clones were obtained and identified by long-range PCR. Four positive homologous recombination clones were obtained. Four positive F1 mice were obtained and confirmed by sequencing. There was no obvious abnormality in the phenotypes of the Xbp1 gene flox-heterozygous mice. Conclusion The conditional knockout flox-heterozygous mouse model of Xbp1 gene was successfully established,which laid a foundation for further studies of the organ or tissue-specific biological effects of Xbp1 gene.
引文
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[2]Glimcher LH.Xbp1:the last two decades[J].Ann Rheum Dis,2010,69(Suppl 1):i67-i71.
[3]Back SH,Schroder M,Lee K,et al.ER stress signaling by regulated splicing:IRE1/HAC1/Xbp1[J].Methods,2005,35(4):395-416.
[4]Lee AH,Iwakoshi NN,Glimcher LH.XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response[J].Mol Cell biol,2003,23(21):7448-7459.
[5]Reimold AM,Etkin A,Clauss I,et al.An essential role in liver development for transcription factor Xbp-1[J].Genes Dev,2000,14(2):152-157.
[6]Reimold AM,Iwakoshi NN,Manis J,et al.Plasma cell differentiation requires the transcription factor Xbp-1[J].Nature,2001,412(6844):300-307.
[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]Heindryckx F,Binet F,Ponticos M,et al.Endoplasmic reticulum stress enhances fibrosis through IRE1alpha-mediated degradation of miR-150 and XBP-1 splicing[J].EMBO Mol Med,2016,8(7):729-744.
[9]郑永.XBP1s在炎症诱导腹膜纤维化形成中的作用[D].第四军医大学,2014.
[10]桑景荭,刘欣.基因敲除小鼠在疾病研究中的应用[J].生物学通报,2009,(12):3-6.
[11]詹振宏,吴伟伟,田月珍,等.基因打靶技术的研究进展[J].中国畜牧兽医,2011,(2):72-75.
[12]石玉衡.基因敲除模型小鼠在疾病研究中的应用[J].中国组织工程研究与临床康复,2011,(33):6239-6242.
[13]Nishihama R,Ishida S,Urawa H,et al.Conditional gene expression/deletion systems for Marchantia polymorpha using its own heat-shock promoter and Cre/loxp-mediated site-specific recombination[J].Plant Cell Physiol,2016,57(2):271-280.
[14]Okuyama T,Isoe Y,Hoki M,et al.Controlled Cre/loxp sitespecific recombination in the developing brain in medaka fish,Oryzias latipes[J].PLo S One,2013,8(6):e66597.
[15]Bouabe H,Okkenhaug K.Gene targeting in mice:a review[J].Methods Mol Biol,2013,1064:315-336.
[16]Pei Z,Chen Q,Koren D,et al.Conditional knock-out of vesicular GABA transporter gene from starburst amacrine cells reveals the contributions of multiple synaptic mechanisms underlying direction selectivity in the retina[J].J Neurosci,2015,35(38):13219-13232.
[17]Li J,Spensberger D,Ahn JS,et al.JAK2 V617F impairs hematopoietic stem cell function in a conditional knock-in mouse model of JAK2 V617F-positive essential thrombocythemia[J].Blood,2010,116(9):1528-1538.
[18]Korpershoek E,Loonen AJ,Corvers S,et al.Conditional Pten knock-out mice:a model for metastatic phaeochromocytoma[J].J Pathol,2009,217(4):597-604.