Neuritin条件性基因打靶载体的构建和同源重组胚胎干细胞克隆鉴定
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摘要
Neuritin(Nrn1)是神经营养因子家族的成员,能够维持神经元的存活和突起的生长,在神经系统发育和再生中起重要作用。为了解Neuritin基因在整体动物水平的系统生物学功能,通过PCR扩增Neuritin基因片段,将该片段用In-fusion无缝克隆技术连接至用限制性内切酶Asc1酶切后线性化的CTVIRES-EGFP质粒,转化至Stellar感受态细胞后,对酶切和测序鉴定正确的阳性克隆子进行大量提取质粒,酶切使之线性化后将其电转导入小鼠胚胎干细胞细胞,经G418筛选,挑取抗性克隆,用PCR方法鉴定出同源重组的ES细胞DNA。结果表明,成功构建了CTV-Nrn1-IRES-EGFP基因打靶载体载体并筛选出阳性同源重组胚胎干细胞,为制备Neuritin条件性基因打靶小鼠模型奠定了实验基础。
Neuritin is a member of the neurotrophic factor family and promotes neuron growth and branching.It has shown to play an important role in the development and regeneration of the nervous system.In order to find the systemic biology function of Neuritin gene from the whole animal level,Neuritin gene fragment was amplified by polymerase chain reaction.The fragment was confirmed by sequencing and then ligated into the CTV-IRES-EGFP plasmid linearized with Asc1 by In-fusion cloning.Substantial extractions of plasmids for the positive clones were picked and identified by plasmid extraction and sequencing.The plasmids were transferred to the mouse ES cells by linearization.ES cells were culture by G418 and the positive homologously recombinant clone was identified by PCR.The result showed that the construction of expression vector CTV-Nrn1-IRES-EGFP was successfully processed and the homologous recombinant ES cells were identified,which will lay an experimental foundation for the preparation of conditioned Neuritin gene targeting mouse models.
Neuritin is a member of the neurotrophic factor family and promotes neuron growth and branching.It has shown to play an important role in the development and regeneration of the nervous system.In order to find the systemic biology function of Neuritin gene from the whole animal level,Neuritin gene fragment was amplified by polymerase chain reaction.The fragment was confirmed by sequencing and then ligated into the CTV-IRES-EGFP plasmid linearized with Asc1 by In-fusion cloning.Substantial extractions of plasmids for the positive clones were picked and identified by plasmid extraction and sequencing.The plasmids were transferred to the mouse ES cells by linearization.ES cells were culture by G418 and the positive homologously recombinant clone was identified by PCR.The result showed that the construction of expression vector CTV-Nrn1-IRES-EGFP was successfully processed and the homologous recombinant ES cells were identified,which will lay an experimental foundation for the preparation of conditioned Neuritin gene targeting mouse models.
引文
[1]Nedivi E,Hevroni D,Naot D,et al.Numerous candidate plasticity-related genes revealed by differential cDNA cloning[J].Nature,1993,363(6431):718-722.
[2]Fujino T,Leslie J H,Eavri R,et al.CPG15regulates synapse stability in the developing and adult brain[J].Genes Dev,2011,25(24):2674-2685.
[3]Han D,Qin B,Liu G,et al.Characterization of neuritin as a novel angiogenic factor[J].Biochem Biophys Res Commun,2011,415(4):608-612.
[4]Marron T U,Guerini V,Rusmini P,et al.Androgen-induced neurite outgrowth is mediated by neuritin in motor neurones[J].J Neurochem,2005,92(1):10-20.
[5]Zito A,Cartelli D,Cappelletti G,et al.Neuritin 1promotes neuronal migration[J].Brain Struct Funct,2014,219(1):105-118.
[6]Yao J J,Zhao Q R,Lu J M,et al.Functions and the related signaling pathways of the neurotrophic factor neuritin[J].Acta Pharmacol Sin,2018,12(8):231-239.
[7]Liu Q,Zhang H,Xu J,et al.Neuritin provides neuroprotection against experimental traumatic brain injury in rats[J].Int J Neurosci,2018,128(9):811-820.
[8]Lu J M,Liu D D,Li Z Y,et al.Neuritin enhances synaptic transmission in medial prefrontal cortex in mice by increasing Cav3.3surface expression[J].Cereb Cortex,2017,27(7):3842-3855.
[9]Cappelletti G,Galbiati M,Ronchi C,et al.Neuritin(cpg15)enhances the differentiating effect of NGF on neuronal PC12cells[J].J Neurosci Res,2007,85(12):2702-2713.
[10]康赟,福林玉,滕艳,等.小鼠Bpi条件基因打靶载体的构建和鉴定[J].实验动物科学,2010,27(4):15-19.
[11]伍志强,韩为东,赵亚力,等.小鼠LRP16基因打靶载体的构建和胚胎干细胞同源重组克隆鉴定[J].中国实验动物学报,2006,14(3):167-171.
[12]Niwa H.Mouse ES cell culture system as a model of development[J].Dev Growth Differ,2010,52(3):275-283.
[13]Divya M S,Rasheed V A,Schmidt T,et al.intraocular injection of ES cell-derived neural progenitors improve visual function in retinal ganglion cell-depleted mouse models[J].Front Cell Neurosci,2017,11(6):295-308.
[14]Lau H T,Liu L,Ray C,et al.Derivation of hybrid ES cell lines from two different strains of mice[J].Stem Cell Res,2016,16(2):252-265.
[15]Liu J L,Yakar S,Leroith D,et al.Conditional knockout of mouse insulin-like growth factor-1gene using the Cre/loxP system[J].Proc Soc Exp Biol Med,2000,223(4):344-351.
[16]Griep A E,John M C,Ikeda S,et al.Gene targeting in the mouse[J].Methods Mol Biol,2011,770(15):293-312.
[17]Jung C J,Zhang J,Trenchard E,et al.Efficient gene targeting in mouse zygotes mediated by CRISPR/Cas9-protein[J].Transgenic Res,2017,26(2):263-277.
[18]Zhou S,Zhou J.Neuritin,a neurotrophic factor in nervous system physiology[J].Curr Med Chem,2014,21(10):1212-1219.
[19]Yao J J,Mei Y A.Progress of study on neuritin[J].Sheng Li Xue Bao,2013,65(5):483-488.
[20]Perez-Campo F M,Spencer H L,Elder R H,et al.Novel vectors for homologous recombination strategies in mouse embryonic stem cells:an ES cell line expressing EGFP under control of the 5T4promoter[J].Exp Cell Res,2007,313(16):3604-3615.
[2]Fujino T,Leslie J H,Eavri R,et al.CPG15regulates synapse stability in the developing and adult brain[J].Genes Dev,2011,25(24):2674-2685.
[3]Han D,Qin B,Liu G,et al.Characterization of neuritin as a novel angiogenic factor[J].Biochem Biophys Res Commun,2011,415(4):608-612.
[4]Marron T U,Guerini V,Rusmini P,et al.Androgen-induced neurite outgrowth is mediated by neuritin in motor neurones[J].J Neurochem,2005,92(1):10-20.
[5]Zito A,Cartelli D,Cappelletti G,et al.Neuritin 1promotes neuronal migration[J].Brain Struct Funct,2014,219(1):105-118.
[6]Yao J J,Zhao Q R,Lu J M,et al.Functions and the related signaling pathways of the neurotrophic factor neuritin[J].Acta Pharmacol Sin,2018,12(8):231-239.
[7]Liu Q,Zhang H,Xu J,et al.Neuritin provides neuroprotection against experimental traumatic brain injury in rats[J].Int J Neurosci,2018,128(9):811-820.
[8]Lu J M,Liu D D,Li Z Y,et al.Neuritin enhances synaptic transmission in medial prefrontal cortex in mice by increasing Cav3.3surface expression[J].Cereb Cortex,2017,27(7):3842-3855.
[9]Cappelletti G,Galbiati M,Ronchi C,et al.Neuritin(cpg15)enhances the differentiating effect of NGF on neuronal PC12cells[J].J Neurosci Res,2007,85(12):2702-2713.
[10]康赟,福林玉,滕艳,等.小鼠Bpi条件基因打靶载体的构建和鉴定[J].实验动物科学,2010,27(4):15-19.
[11]伍志强,韩为东,赵亚力,等.小鼠LRP16基因打靶载体的构建和胚胎干细胞同源重组克隆鉴定[J].中国实验动物学报,2006,14(3):167-171.
[12]Niwa H.Mouse ES cell culture system as a model of development[J].Dev Growth Differ,2010,52(3):275-283.
[13]Divya M S,Rasheed V A,Schmidt T,et al.intraocular injection of ES cell-derived neural progenitors improve visual function in retinal ganglion cell-depleted mouse models[J].Front Cell Neurosci,2017,11(6):295-308.
[14]Lau H T,Liu L,Ray C,et al.Derivation of hybrid ES cell lines from two different strains of mice[J].Stem Cell Res,2016,16(2):252-265.
[15]Liu J L,Yakar S,Leroith D,et al.Conditional knockout of mouse insulin-like growth factor-1gene using the Cre/loxP system[J].Proc Soc Exp Biol Med,2000,223(4):344-351.
[16]Griep A E,John M C,Ikeda S,et al.Gene targeting in the mouse[J].Methods Mol Biol,2011,770(15):293-312.
[17]Jung C J,Zhang J,Trenchard E,et al.Efficient gene targeting in mouse zygotes mediated by CRISPR/Cas9-protein[J].Transgenic Res,2017,26(2):263-277.
[18]Zhou S,Zhou J.Neuritin,a neurotrophic factor in nervous system physiology[J].Curr Med Chem,2014,21(10):1212-1219.
[19]Yao J J,Mei Y A.Progress of study on neuritin[J].Sheng Li Xue Bao,2013,65(5):483-488.
[20]Perez-Campo F M,Spencer H L,Elder R H,et al.Novel vectors for homologous recombination strategies in mouse embryonic stem cells:an ES cell line expressing EGFP under control of the 5T4promoter[J].Exp Cell Res,2007,313(16):3604-3615.