带有雌激素受体编码区的KLF4表达载体的构建及其转染后对细胞的可调控表达
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摘要
目的构建KLF4-ER基因表达的带有加强型绿色荧光蛋白(EGFP)的博来霉素抗性的pCGF5 IEGZ载体,利用其对小鼠巨噬细胞系RAW264.7进行稳定转染株的筛选并鉴定。方法以pEGFP-KLF4质粒为模板,设计带有酶切位点的KLF4上下游引物,以PCR扩增获得编码序列,其产物电泳鉴定后纯化回收,并将改良型小鼠雌激素受体的激素链接区序列与之融合,KLF4-ER片段纯化回收后插入pCGF5-IEGZ载体中;重组体转化细菌,筛选扩增后酶切鉴定;重组质粒转染细胞,倒置荧光显微镜观察EGFP的表达,使用zeocin筛选稳定株,流式细胞术检测转染率;Western Blot、RT-PCR检测转染后的KLF4表达;经Tamoxifen诱导后,DAPI核染色观察重组载体转运胞核中的表达以及抗酒石酸酸性磷酸酶(TRAP)染色检测目的基因表达的效应。结果电泳鉴定KLF4全长片段及pCGF5 IEGZ-KLF4ER重组体双酶切结果符合设定要求,重组质粒构建成功;目的载体成功转入细胞中,转染19 d后流式细胞测定稳定株转染效率达95.31%;20 d KLF4蛋白及mRNA水平均明显高于空转组和阴性组(P<0.01)。Tamoxifen诱导靶基因的表达生物效应优于对照组。结论该实验成功地用pCGF5 IEGZ型载体将带有可受调控的雌激素受体编码区的目的基因导入通用的小鼠巨噬细胞系中,为今后研究靶基因在破骨细胞中可诱导性过表达提供了新的途径。
Objective To construct pCGF5 IEGZ-KLF4-ER eukaryotic expression vector containing enhanced green fluorescent protein(EGFP)and zeocin resistance gene and to screen and identification the recombinant transfected murine RAW264.7 macrophages.Methods The pEGFP-KLF4 was used as templates,KLF4 oligonucleotide primers were designed(containing the both enzyme site) to amplify the coding sequence.The PCR product was electrophoresed and purified,a modified ligand-binding domain construct of murine oestrogen receptor(MOR G525R) was fused to KLF4,and then inserted into the pCGF5-IEGZ vector.The recombinant was transformed into DH5α.E.coli.and then appraised by digestion with restriction endonuclease.The optimum dose of zeocin for the RAW264.7 kill was chose to screen the transfected cells.Transfection efficiency was determined by using flow cytometry at 19d after transfection,the expression of KLF4 was detected by Western Blot and RT-PCR.localization of the recombinant in cells was determined after DPAI staining.Observed the effect of KLF4 on osteoclast differentiation induced by Tamoxifen.Results Both the lengths of KLF4 PCR product and the KLF4-ER gene fragment form recombinant were consistent with expected,expression vector construction was achieved.Transfection efficiency was 95.31% 19d after transfection,the expression of both KLF4 protein and mRNA were greater and higher than pCGF5 IEGZ and non-transfected groups.The biological effects of KLF4 induced by Tamoxifen were better.Conclusion The recombinant expression vector pCGF5 IEGZ-KLF4ER has been successfully established,it was the first domestic application of pCGF5 IEGZ vector containing inducible HBD of ER for RAW264.7 transfection,and we wish it would provide a new method for the research of regulated overexpression of target gene in osteoclast.
Objective To construct pCGF5 IEGZ-KLF4-ER eukaryotic expression vector containing enhanced green fluorescent protein(EGFP)and zeocin resistance gene and to screen and identification the recombinant transfected murine RAW264.7 macrophages.Methods The pEGFP-KLF4 was used as templates,KLF4 oligonucleotide primers were designed(containing the both enzyme site) to amplify the coding sequence.The PCR product was electrophoresed and purified,a modified ligand-binding domain construct of murine oestrogen receptor(MOR G525R) was fused to KLF4,and then inserted into the pCGF5-IEGZ vector.The recombinant was transformed into DH5α.E.coli.and then appraised by digestion with restriction endonuclease.The optimum dose of zeocin for the RAW264.7 kill was chose to screen the transfected cells.Transfection efficiency was determined by using flow cytometry at 19d after transfection,the expression of KLF4 was detected by Western Blot and RT-PCR.localization of the recombinant in cells was determined after DPAI staining.Observed the effect of KLF4 on osteoclast differentiation induced by Tamoxifen.Results Both the lengths of KLF4 PCR product and the KLF4-ER gene fragment form recombinant were consistent with expected,expression vector construction was achieved.Transfection efficiency was 95.31% 19d after transfection,the expression of both KLF4 protein and mRNA were greater and higher than pCGF5 IEGZ and non-transfected groups.The biological effects of KLF4 induced by Tamoxifen were better.Conclusion The recombinant expression vector pCGF5 IEGZ-KLF4ER has been successfully established,it was the first domestic application of pCGF5 IEGZ vector containing inducible HBD of ER for RAW264.7 transfection,and we wish it would provide a new method for the research of regulated overexpression of target gene in osteoclast.
引文
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[9]McConnell BB,Ghaleb AM,Nandan MO,et al.Thediverse functions of Krüpple-like factors 4 and 5 inepithelial biology and pathobiology.Bioessays,2007,29(6):549-557.
[10]Liao X,Sharma N,Kapadia F,et al.Krüppel-likefactor 4 regulates macrophage polarization.J Clin In-vest,2011,121(7):2736-2749.
[11]Liu J,Liu Y,Zhang H,et al.KLF4 promotes theexpression,translocation,and release of HMGB1 inRAW264.7 macrophages in response to LPS.Shock,2008,30(3):260-266.
[12]Nakashima T,Takayanagi H.New regulation mech-anisms of osteoclast differentiation.Ann N Y AcadSci,2011,1240:E13-18.
[13]王钢,李亚明,刘世清,等.人工关节磨损钛微粒诱导破骨的RANK/OPG通路机制的实验研究.生物骨科材料与临床研究,2010,7(5):6-9.
[14]Chen Y,Rabson AB,Gorski DH.et al.MEOX2regulates nuclear factor-kappaB activity in vascularendothelial cells through interactions with p65 andIkappaBbeta.Cardiovasc Res,2010,87(4):723-731.
[15]Lindemann D,Patriquin E,Feng S,et al.Versatileretrovirus vector systems for regulated gene expres-sion in vitro and in vivo.Mol Med,1997,3(7):466-476.
[16]Odegaard JI,Ricardo-Gonzalez RR,Goforth MH,etal.Macrophage-specific PPARgamma controls alter-native activation and improves insulin resistance.Na-ture,2007,447(7148):1116-1120.
[2]Alder JK,Georgantas RW,Hildreth RL,et al.Kruppel-like factor 4 is essential for inflammatorymonocyte differentiation in vivo.J Immunol,2008,180(8):5645-5652.
[3]Feinberg MW,Wara AK,Cao Z,et al.TheKrüppel-like factor KLF4 is a critical regulator ofmonocyte differentiation.EMBO J,2007,26(18):4138-4148.
[4]Feinberg MW,Cao Z,Wara AK,et al.Kruppel-likefactor 4 is a mediator of proinflammatory signaling inmacrophages.J Biol Chem,2005,280(46):38247-3858.
[5]Liu J,Yang T,Liu Y,et al.Krüppel-like factor 4inhibits the expression of interleukin-1 beta in lipopo-lysaccharide-induced RAW264.7 macrophages.FEBSLett,2012,586(6):834-840.
[6]Littlewood TD,Hancock DC,Danielian PS,et al.Amodified oestrogen receptor ligand-binding domain asan improved switch for the regulation of heterologousproteins.Nucleic Acids Res,1995,23(10):1686-1690.
[7]Oates AC,Pratt SJ,Vail B,et al.The zebrafish klfgene family.Blood,2001,98(6):1792-1801.
[8]Rowland BD,Peeper DS.KLF4,p21 and context-de-pendent opposing forces in cancer.Nat Rev Cancer,2006,6(1):11-23.
[9]McConnell BB,Ghaleb AM,Nandan MO,et al.Thediverse functions of Krüpple-like factors 4 and 5 inepithelial biology and pathobiology.Bioessays,2007,29(6):549-557.
[10]Liao X,Sharma N,Kapadia F,et al.Krüppel-likefactor 4 regulates macrophage polarization.J Clin In-vest,2011,121(7):2736-2749.
[11]Liu J,Liu Y,Zhang H,et al.KLF4 promotes theexpression,translocation,and release of HMGB1 inRAW264.7 macrophages in response to LPS.Shock,2008,30(3):260-266.
[12]Nakashima T,Takayanagi H.New regulation mech-anisms of osteoclast differentiation.Ann N Y AcadSci,2011,1240:E13-18.
[13]王钢,李亚明,刘世清,等.人工关节磨损钛微粒诱导破骨的RANK/OPG通路机制的实验研究.生物骨科材料与临床研究,2010,7(5):6-9.
[14]Chen Y,Rabson AB,Gorski DH.et al.MEOX2regulates nuclear factor-kappaB activity in vascularendothelial cells through interactions with p65 andIkappaBbeta.Cardiovasc Res,2010,87(4):723-731.
[15]Lindemann D,Patriquin E,Feng S,et al.Versatileretrovirus vector systems for regulated gene expres-sion in vitro and in vivo.Mol Med,1997,3(7):466-476.
[16]Odegaard JI,Ricardo-Gonzalez RR,Goforth MH,etal.Macrophage-specific PPARgamma controls alter-native activation and improves insulin resistance.Na-ture,2007,447(7148):1116-1120.