一种组织特异性Connexin31表达载体的构建
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摘要
背景:
间隙连接蛋白(Connexins)是一类可以在细胞的膜上组装成为间隙连接通道(Gap Junctions)以介导相邻细胞间小分子物质(＜1000Da)的重要蛋白家族。通过这些物质和信息的交换,使得细胞间对外界刺激作出协调一致的反应。
Connexin31(CX31)是间隙连接蛋白家族的一员,其突变可导致显、隐性红皮肤角化病(dominant and/recessive EKV),显、隐性神经性耳聋(dominantand/recessive hearing impairment),或者显性神经性耳聋-周围神经病(dominanthearing impairment combined with peripheral neuropathy)等。CX31的基因敲除小鼠中没有发现耳聋或皮肤病的表型,以人CX31蛋白为启动子的转基因小鼠研究发现其F137L突变体小鼠表皮可出现部分角质化。CX31对于细胞稳态维持、细胞增殖、分化具有重要作用。但对CX31通道组装、通道通透性、通道功能调节和细胞内运输等CX31分子特征和细胞行为的认识还很粗略。
目的:
增强CX31在细胞中的表达水平,尤其是突变体在某一特定组织中的表达水平,将有可能使动物模型出现相应的表型。本研究用Keratin14蛋白启动子构建CX31野生型和F137L突变体的表达载体,并研究该载体的皮肤组织表达能力和表达特异性。得到一种可望用于转基因动物模型的皮肤组织特异性表达的CX31载体。
方法:
我们使用PCR技术,将CX31产物连接到,含有Keratin14启动子、兔globin和Keratin14的3'非翻译区的载体中。然后将该载体用Lipifectamine LTX转染Hacat细胞,应用免疫沉淀和免疫荧光技术对CX31的表达进行检测。为了研究其组织表达的特异性,我们还用磷酸钙法转染了HEK 293T细胞,并用免疫荧光技术对CX31的表达情况进行了检测。
结果:
通过酶切及测序证实我们得到了CX31野生型和F137L突变体载体;Hacat细胞的免疫沉淀和免疫荧光检测到了CX31的表达,且野生型CX31可以形成间隙连接斑(gap junction plaques),F137L突变的蛋白则不能;对HEK293T细胞的免疫荧光检测没有发现以Keratin14启动子的载体的CX31的表达,而转染了pCDNA3.1A(-)-CX31-myc的HEK 293T细胞阳性对照中则能够通过免疫荧光检测到蛋白表达和形成的间隙连接斑。
结论:
本研究构建的CX31组织特异性表达载体能有效表达,在线性化去除其部分骨架后可望用于转基因动物模型的构建。
Background
Connexins is a protein family that can form a Gap Junction structure on the membrane and allows the exchange of small molecules(＜1kD) between cells.The cells then synchronize their response of foreign stimulus.
Connexin31(CX31)is one member of the Connexin family which its mutations are found to cause dominant or recessive EKV,dominant or recessive hearing impairment and dominant hearing impairment combined with peripheral neuropathy.There is no hearing loss or skin disease phenotype found in CX31 knockout mouse and a research which created a transgenic mouse in using of human CX31 promoter found partially keratinise of the epidermis.CX31 plays very important role in cell stability,proliferation and differentiation.But we still know little of CX31 character and activity in Gap Junction assembling,permeability, function regulation and intra-cellular trafficking.
Research goals
To enhance the expression level of CX31,especially some mutation's expression in specific tissue may cause the phenotype in animal models.We employed a Keratin 14 promoter and made an expression construct of CX31 wt and F137L mutant,then studied its expression ability and specificity in skin sourced cell line.We obtain a skin tissue specifically expressed construct which also can be used to create a transgenic animal model.
Methods
We use PCR to amplify CX31 wt and F137L mutant and ligate it into a vector contains Keratin14 promoter,rabbit globin and Keratin 14 3' Untanslated region elements.Thereafter,tansfect the constructs in to Hacat cell use Lipofectamine LTX and detect the CX31 with immunoprecipitation and immunostaining.We also transfect the HEK 293T cell by calcium phosphate method and detect its expression level by immunostaining in order to evaluate the ability of tissue specific expression.
Results:
The constructs are established by enzyme digestion and sequencing. The expression in Hacat cell is detected by immunoprecipitation and immunostaining,which the CX31 wild type protein can form the Gap Junction plaques while the F137L mutant can not.We do not detect the CX31 expression in HEK 293T cells by using Keratinl4 promoter,but the positive control which was transfected with pCDNA3.1A(-)-CX31-myc has the expression and Gap Junction plaques.
Conclusions
The two constructs made in this research can successfully and specifically express in skin tissue sourced cell line.They also are available in the creation of transgenic animal model after linearization to remove their vector backbones.
间隙连接蛋白(Connexins)是一类可以在细胞的膜上组装成为间隙连接通道(Gap Junctions)以介导相邻细胞间小分子物质(＜1000Da)的重要蛋白家族。通过这些物质和信息的交换,使得细胞间对外界刺激作出协调一致的反应。
Connexin31(CX31)是间隙连接蛋白家族的一员,其突变可导致显、隐性红皮肤角化病(dominant and/recessive EKV),显、隐性神经性耳聋(dominantand/recessive hearing impairment),或者显性神经性耳聋-周围神经病(dominanthearing impairment combined with peripheral neuropathy)等。CX31的基因敲除小鼠中没有发现耳聋或皮肤病的表型,以人CX31蛋白为启动子的转基因小鼠研究发现其F137L突变体小鼠表皮可出现部分角质化。CX31对于细胞稳态维持、细胞增殖、分化具有重要作用。但对CX31通道组装、通道通透性、通道功能调节和细胞内运输等CX31分子特征和细胞行为的认识还很粗略。
目的:
增强CX31在细胞中的表达水平,尤其是突变体在某一特定组织中的表达水平,将有可能使动物模型出现相应的表型。本研究用Keratin14蛋白启动子构建CX31野生型和F137L突变体的表达载体,并研究该载体的皮肤组织表达能力和表达特异性。得到一种可望用于转基因动物模型的皮肤组织特异性表达的CX31载体。
方法:
我们使用PCR技术,将CX31产物连接到,含有Keratin14启动子、兔globin和Keratin14的3'非翻译区的载体中。然后将该载体用Lipifectamine LTX转染Hacat细胞,应用免疫沉淀和免疫荧光技术对CX31的表达进行检测。为了研究其组织表达的特异性,我们还用磷酸钙法转染了HEK 293T细胞,并用免疫荧光技术对CX31的表达情况进行了检测。
结果:
通过酶切及测序证实我们得到了CX31野生型和F137L突变体载体;Hacat细胞的免疫沉淀和免疫荧光检测到了CX31的表达,且野生型CX31可以形成间隙连接斑(gap junction plaques),F137L突变的蛋白则不能;对HEK293T细胞的免疫荧光检测没有发现以Keratin14启动子的载体的CX31的表达,而转染了pCDNA3.1A(-)-CX31-myc的HEK 293T细胞阳性对照中则能够通过免疫荧光检测到蛋白表达和形成的间隙连接斑。
结论:
本研究构建的CX31组织特异性表达载体能有效表达,在线性化去除其部分骨架后可望用于转基因动物模型的构建。
Background
Connexins is a protein family that can form a Gap Junction structure on the membrane and allows the exchange of small molecules(＜1kD) between cells.The cells then synchronize their response of foreign stimulus.
Connexin31(CX31)is one member of the Connexin family which its mutations are found to cause dominant or recessive EKV,dominant or recessive hearing impairment and dominant hearing impairment combined with peripheral neuropathy.There is no hearing loss or skin disease phenotype found in CX31 knockout mouse and a research which created a transgenic mouse in using of human CX31 promoter found partially keratinise of the epidermis.CX31 plays very important role in cell stability,proliferation and differentiation.But we still know little of CX31 character and activity in Gap Junction assembling,permeability, function regulation and intra-cellular trafficking.
Research goals
To enhance the expression level of CX31,especially some mutation's expression in specific tissue may cause the phenotype in animal models.We employed a Keratin 14 promoter and made an expression construct of CX31 wt and F137L mutant,then studied its expression ability and specificity in skin sourced cell line.We obtain a skin tissue specifically expressed construct which also can be used to create a transgenic animal model.
Methods
We use PCR to amplify CX31 wt and F137L mutant and ligate it into a vector contains Keratin14 promoter,rabbit globin and Keratin 14 3' Untanslated region elements.Thereafter,tansfect the constructs in to Hacat cell use Lipofectamine LTX and detect the CX31 with immunoprecipitation and immunostaining.We also transfect the HEK 293T cell by calcium phosphate method and detect its expression level by immunostaining in order to evaluate the ability of tissue specific expression.
Results:
The constructs are established by enzyme digestion and sequencing. The expression in Hacat cell is detected by immunoprecipitation and immunostaining,which the CX31 wild type protein can form the Gap Junction plaques while the F137L mutant can not.We do not detect the CX31 expression in HEK 293T cells by using Keratinl4 promoter,but the positive control which was transfected with pCDNA3.1A(-)-CX31-myc has the expression and Gap Junction plaques.
Conclusions
The two constructs made in this research can successfully and specifically express in skin tissue sourced cell line.They also are available in the creation of transgenic animal model after linearization to remove their vector backbones.
引文
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1.Richard,G.,Smith,L.E.,Bailey,R.A.,et al.,Mutations in the human connexin gene GJB3 cause erythrokeratodermia variabilis.Nat Genet,1998.20(4):366-9
2.Paul,D.L.,Molecular cloning of cDNA for rat liver gap junction protein.J Cell Biol,1986,103(1):123-34
3.Kumar,N.M.,Gilula,N.B.,Cloning and characterization of human and rat liver cDNAs coding for a gap junction protein.J Cell Biol,1986,103(3):767-76
4.Gottfried,I.,Landau,M.,Glaser,F.,et al.,A mutation in GJB3is associated with recessive erythrokeratodermia variabilis(EKV)and leads to defective trafficking of the connexin 31 protein.Hum Mol Genet,2002.11(11):1311-6
5.Xia,J.H.,Liu,C.Y.,Tang,B.S.,et al.,Mutations in the gene encoding gap junction protein beta-3 associated with autosomal dominant hearing impairment.Nat Genet,1998.20(4):370-3
6.Liu,X.Z.,Xia,X.J.,Xu,L.R.,et al.,Mutations in connexin31underlie recessive as well as dominant non-syndromic hearing loss. Hum Mol Genet, 2000. 9(1): 63-7
7. Richard, G., Brown, N., Smith, L.E., et al., The spectrum of mutations in erythrokeratodermias-novel and de novo mutations in GJB3. Hum Genet, 2000. 106(3): 321-9
8. Rabionet, R, Gasparini, P., et al., Molecular Genetics of hearing impairment due to mutations in gap junction genes encoding beta connexins. Human Mutation 2000, 16:190-202
9. Hoh, J. H., John, S. A. and Revel, J. P., Molecular cloning and characterization of a new member of the gap junction gene family, connexin-31. J Biol Chem, 1991. 266(10): 6524-31
10. Wenzel, K., Manthey, D., Willecke, K., et al., Human gap junction protein connexin31: molecular cloning and expression analysis. Biochem Biophys Res Commun, 1998, 248(3): 910-5
11. Reuss, B., Hellmann, P., Traub, O., et al., Expression of connexin31 and connexin43 genes in early rat embryos. Dev Genet, 1997. 21(1): 82-90
12. Plum, A., Winterhager, E., Pesch, J., et al., Connexin31-deficiency in mice causes transient placental dysmorphogenesis but does not impair hearing and skin differentiation. Dev Biol, 2001. 231(2): 334-47
13. Di, W. L., Rugg, E. L., Leigh, I. M., et al., Multiple epidermal connexins are expressed in different keratinocyte subpopulations including Connexin 31. J Invest Dermatol, 2001. 117(4): 958-64
14. Tucker, M. A., Barajas, L., Rat connexins 30. 3 and 31 are expressed in the kidney. Exp Cell Res, 1994, 213(1): 224-30
15. Gabriel, H. D., Strobl, B., Hellmann, P., et al., Organization and regulation of the rat CX31 gene. Implication for a crucial role of the intron region. Eur J Biochem, 2001. 268(6): 1749-59
16. Jungbluth, S., Willecke, K., Champagnat, J., Segment-specific expression of connexin31 in the embryonic hindbrain is regulated by Krox20.Dev Dyn,2002.223(4):544-51
17.Heinz-Dieter Gabriel,Barabara Strobl,Peter Hellmann,et al.,Organization and regulation of the rat CX31 gene.Eur J.Biochem,2001,268(6):6524-6531
18.AchimPlum,G.H.,Willeche,K.,Expression of the Mouse Gap Junction Gene Gjb3 Is Regulated by Distinct Mechanisms in Embryonic Stem Cells and Keratinocytes.Genomics,79(1):24-30
19.Butterweck,A.,Elfgang,C.,Willecke,K.,et al.,Differential expression of the gap junction proteins connexin45,-43,-40,-31,and -26 in mouse skin.Eur J Cell Biol,1994,65(1):152-63
20.Lopez-Bigas,N.,Olive,M.,Rabionet,R.,et al.,Connexin 31(GJB3)is expressed in the peripheral and auditory nerves and causes neuropathy and hearing impairment.Hum Mol Genet,2001.10(9):947-52
21.张宗磊.Connxin 31间隙连接通道功能调节研究:[硕士学位论文].长沙:中南大学,2007
22.Diestel,S.,Richard,G.,Doring,B.,et al.,Expression of a connexin31 mutation causing erythrokeratodermia variabilis is lethal for HeLa cells.Biochem Biophys Res Commun,2002,296(3):721-8
23.Gottfried,I.,Landau,M.,Glaser,F.,et al.,A mutation in GJB3is associated with recessive erythrokeratodermia variabilis(EKV)and leads to defective trafficking of the connexin 31 protein.Human Molecular Genetics,2002,11(11):1311-1316
24.Rouan,F.,Lo,C.W.,Fertala,A.,et al.,Divergent effects of two sequence variants of GJB3(G12D and R32W)on the function of connexin 31 in vitro.Exp Dermatol,2003,12(2):191-7
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2.Paul,D.L.,Molecular cloning of cDNA for rat liver gap junction protein.J Cell Biol,1986,103(1):123-34
3.Kumar,N.M.,Gilula,N.B.,Cloning and characterization of human and rat liver cDNAs coding for a gap junction protein.J Cell Biol,1986,103(3):767-76
4.Gottfried,I.,Landau,M.,Glaser,F.,et al.,A mutation in GJB3is associated with recessive erythrokeratodermia variabilis(EKV)and leads to defective trafficking of the connexin 31 protein.Hum Mol Genet,2002.11(11):1311-6
5.Xia,J.H.,Liu,C.Y.,Tang,B.S.,et al.,Mutations in the gene encoding gap junction protein beta-3 associated with autosomal dominant hearing impairment.Nat Genet,1998.20(4):370-3
6.Liu,X.Z.,Xia,X.J.,Xu,L.R.,et al.,Mutations in connexin31underlie recessive as well as dominant non-syndromic hearing loss. Hum Mol Genet, 2000. 9(1): 63-7
7. Richard, G., Brown, N., Smith, L.E., et al., The spectrum of mutations in erythrokeratodermias-novel and de novo mutations in GJB3. Hum Genet, 2000. 106(3): 321-9
8. Rabionet, R, Gasparini, P., et al., Molecular Genetics of hearing impairment due to mutations in gap junction genes encoding beta connexins. Human Mutation 2000, 16:190-202
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10. Wenzel, K., Manthey, D., Willecke, K., et al., Human gap junction protein connexin31: molecular cloning and expression analysis. Biochem Biophys Res Commun, 1998, 248(3): 910-5
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12. Plum, A., Winterhager, E., Pesch, J., et al., Connexin31-deficiency in mice causes transient placental dysmorphogenesis but does not impair hearing and skin differentiation. Dev Biol, 2001. 231(2): 334-47
13. Di, W. L., Rugg, E. L., Leigh, I. M., et al., Multiple epidermal connexins are expressed in different keratinocyte subpopulations including Connexin 31. J Invest Dermatol, 2001. 117(4): 958-64
14. Tucker, M. A., Barajas, L., Rat connexins 30. 3 and 31 are expressed in the kidney. Exp Cell Res, 1994, 213(1): 224-30
15. Gabriel, H. D., Strobl, B., Hellmann, P., et al., Organization and regulation of the rat CX31 gene. Implication for a crucial role of the intron region. Eur J Biochem, 2001. 268(6): 1749-59
16. Jungbluth, S., Willecke, K., Champagnat, J., Segment-specific expression of connexin31 in the embryonic hindbrain is regulated by Krox20.Dev Dyn,2002.223(4):544-51
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20.Lopez-Bigas,N.,Olive,M.,Rabionet,R.,et al.,Connexin 31(GJB3)is expressed in the peripheral and auditory nerves and causes neuropathy and hearing impairment.Hum Mol Genet,2001.10(9):947-52
21.张宗磊.Connxin 31间隙连接通道功能调节研究:[硕士学位论文].长沙:中南大学,2007
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23.Gottfried,I.,Landau,M.,Glaser,F.,et al.,A mutation in GJB3is associated with recessive erythrokeratodermia variabilis(EKV)and leads to defective trafficking of the connexin 31 protein.Human Molecular Genetics,2002,11(11):1311-1316
24.Rouan,F.,Lo,C.W.,Fertala,A.,et al.,Divergent effects of two sequence variants of GJB3(G12D and R32W)on the function of connexin 31 in vitro.Exp Dermatol,2003,12(2):191-7
25.Plantard,L.,Huber,M.,Macari,F.,et al.,Molecular interaction of Connexin 30.3 and Connexin 31 suggests a dominant-negative mechanism associated with erythrokeratodermia variabilis. Hum Mol Genet. 2003, 12(24):3287-94
26. Schnichels, M., Worsdorfer, P., et al., The connexin31 F137L mutant mouse as a model for the human skin disease erythrokeratodermia variabilis (EKV). Hum Mol Genet, 2007, 16(10):1216-24
27. Kretz, M., Euwens, C., Hombach, S., et al., Altered Connexin expression and wound healing in the epidermis of connexin-deficient mice. J Cell Sci, 2003, 116(16): 3443-52
28. Diestel, S., Eckert, R., Hulser, D., et al., Exchange of serine residues 263 and 266 reduces the function of mouse gap junction protein connexin31 and exhibits a dominant -negative effect on the wild-type protein in HeLa cells. Exp Cell Res, 2004, 294(2) :446-57