四环素诱导的Cre重组酶表达载体的构建及其体外表达研究
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摘要
Cre/loxP系统介导的条件性基因敲除(conditional gene knockout)克服了传统基因敲除的诸多弊端,是基因功能研究的最重要的技术之一。在此基础上,Cre /loxP系统与Tet基因表达系统相结合建立起的诱导性条件性基因敲除技术,能够更精确地研究基因的功能。该技术需要制备两种遗传工程小鼠,一种是时间特异性可诱导表达Cre的转基因小鼠,另一种是目的基因片段两侧带有同向loxP位点的基因打靶小鼠,通过两种鼠系的杂交,可实现可诱导性的时间特异性的基因敲除。
本研究构建和鉴定了两个诱导表达Cre的真核表达载体,并对这两个载体的转录活性进行了初步研究,为建立诱导性表达Cre的转基因小鼠及进一步制备条件性ADAM10基因敲除的小鼠奠定了基础,有助于开展ADAM10基因功能的研究。采用分子生物学技术,将Tet-On基因表达系统的pTRE载体线性化,并与pCre-IRES-EGFP质粒中长约2.7kb的片段连接,构建成重组质粒pTRE-Cre-EGFP(P1)。将P1与Tet-On基因表达系统中的另一载体pTet-On共转染293T细胞,加入或者不加强力霉素(Doxycycline,Dox),观察绿色荧光和Cre重组酶基因的表达。切下P1载体的部分片段Phcmv-Cre-EGFP与pTet-On载体的部分片段Pcmv-rtTA连接成Pcmv-rtTA-EGFP-Cre-Phcmv(P2)载体,将其转染入293T细胞,加入或者不加Dox诱导,对照观察绿色荧光和Cre重组酶基因的表达。P1与pTet-On的共转染及P2载体的单转染均以pCre-IRES-EGFP为阳性对照组,分别对诱导表达出的Cre重组酶基因进行RT-PCR鉴定。
结果显示,经酶切和测序鉴定,成功构建了P1和P2载体。采用脂质体LP2000将P1与pTet-On质粒共转染293T细胞后,不加Dox时,没有或者很少表达绿色荧光;阳性对照组表达绿色荧光。而加入Dox后,P1与pTet-On质粒共转染的细胞诱导表达出绿色荧光;转染48小时后,分别提取其RNA,经RT-PCR检测表明,诱导表达出绿色荧光的细胞,均能检测出Cre基因的表达;反之,则检测不出Cre基因的表达,证实了绿色荧光和Cre基因的表达是一致的。
采用LP2000将P2转染293T细胞后,不加Dox时,没有绿色荧光的表达,阳性对照组表达绿色荧光,而加入Dox后,P2的转染能诱导表达绿色荧光;转染48小时后分别提取其RNA进行RT-PCR检测,诱导表达出绿色荧光时均能从mRNA水平上检测出Cre基因的表达;反之,则检测不出Cre基因的表达。
综上所述,得出以下结论:
本研究成功构建并鉴定了诱导性表达Cre重组酶的载体(P1和P2)。P1和pTet-On质粒的共转染能在293T细胞中诱导表达绿色荧光,并能从mRNA水平上检测出Cre基因的表达。P2的转染也能在293T细胞中诱导表达绿色荧光,并能从mRNA水平上检测出Cre基因的表达。实验结果证明,本研究所构建的诱导性表达Cre的载体P1和P2均能以时间特异性的方式诱导表达Cre重组酶基因,从细胞水平上证实了P1和P2载体的基因元件在体外均能发挥正常的功能,可以用于制备诱导性表达Cre重组酶的转基因小鼠。而且,使用P2载体仅需制备一种转基因小鼠就能满足研究的需要。
Conditional gene knockout mediated by Cre/loxP system is one of the most important technologies for the study of gene function.It circumvented some limitations of conventional knockout technology.On its basis , the inducible conditional gene knockout technology which combinates Tet gene expression system with Cre / loxP system can be used to study the function of genes more precisely.This technology requires two types of genetically engineered mice,one type is transgenic mice which can express Cre recombinase in the inducible manner,the other is gene targeting mice with loxP sites on both sides of targeting gene fragments.By the cross breeding of these two mouse lines, the inducible time-specific gene knockout can be achieved.
In this study two eukaryotic expression vectors were constructed and identified.They can inducibly express Cre recombinase. And the transcription activity of the vectors was also studied. This study lays the foundation for the generation of the mice inducibly expressing Cre and the conditional ADAM10 gene knockout mice, which will be helpful to understand the function of ADAM 10 gene.
By using the molecular biology technology, pTRE vectors of the Tet-On Gene Expression System were linearized and ligated to a 2.7kb fragments from the plasmid pCre-IRES-EGFP, as a result, the recombinant plasmid pTRE-Cre-EGFP (P1)was constructed.Then P1 and the pTet-On plasmid which was also from the Tet-On Gene Expression system were cotransfected into 293T cells with or without Dox, we observed whether or not there would be the green fluorescence and the expression of Cre recombinase. Phcmv-Cre-EGFP DNA fragments from the P1 vectors were ligated to Pcmv-rtTA fragments from the pTet-On vectors to form the vector P2. Then P2 vectors was transfected into 293T cells with or without Dox, we also observed green fluorescence and the expression of Cre recombinase, respectively.Plasmid pCre-IRES-EGFP was used as a positive control,either in the cotransfection of P1 and pTet-On or in the transfection of P2. And the inducible expression of Cre recombinase was identified by RT-PCR.
Result: Restriction endonuclease digestion and sequencing showed that vector P1 and P2 were successfully constructed.After P1 and pTet-On plasmid were cotransfected into 293T cells mediated by LP2000, without the existence of Dox,there was little or no expression of green fluorescence,while the positive control group expressed green fluorescence. after dox was added, the cotransfection of P1 and the pTet-On plasmid could induce the expression of green fluorescence. 48 hours after the transfection, RNAs were extracted for RT-PCR detection, respectively. The expression of Cre gene can be detected at mRNA levels, so long as the green fluorescence was induced.Otherwise, there was no expression of Cre gene.After P2 was transfected into 293T cells mediated by LP2000, without the Dox,there was little or no expression of green fluorescence.Only the positive control group expressed green fluorescence. But when dox was added, the transfection of P2 could induce the expression of green fluorescence. 48 hours after the transfection, RNAs were extracted for RT-PCR detection. When green fluorescence was induced, the expression of Cre gene can be detected at the mRNA level.Or there was no expression of Cre gene.
In conclusion, we successfully constructed and identified vectors that can inducibly express Cre recombinase (P1 and P2). The cotransfection of P1 and the pTet-On plasmid can induce the expression of green fluorescence in 293T cells.And the transfection of P2 can also induce the expression of green fluorescence in 293T cells.And the expression of Cre gene can be detected at the mRNA level. The results prove that the vector P1 and P2 we constructed induce the expression of Cre recombinase gene in time-specific manner, confirming that the gene elements in the vector P1 and P2 play a normal role in the cells. Both vector P1 and P2 can be used to prepare the Cre transgenic mouse. Moreover, we only prepare a kind of transgenic mice with the vector P2, which will satisfy the requirements for our research.
本研究构建和鉴定了两个诱导表达Cre的真核表达载体,并对这两个载体的转录活性进行了初步研究,为建立诱导性表达Cre的转基因小鼠及进一步制备条件性ADAM10基因敲除的小鼠奠定了基础,有助于开展ADAM10基因功能的研究。采用分子生物学技术,将Tet-On基因表达系统的pTRE载体线性化,并与pCre-IRES-EGFP质粒中长约2.7kb的片段连接,构建成重组质粒pTRE-Cre-EGFP(P1)。将P1与Tet-On基因表达系统中的另一载体pTet-On共转染293T细胞,加入或者不加强力霉素(Doxycycline,Dox),观察绿色荧光和Cre重组酶基因的表达。切下P1载体的部分片段Phcmv-Cre-EGFP与pTet-On载体的部分片段Pcmv-rtTA连接成Pcmv-rtTA-EGFP-Cre-Phcmv(P2)载体,将其转染入293T细胞,加入或者不加Dox诱导,对照观察绿色荧光和Cre重组酶基因的表达。P1与pTet-On的共转染及P2载体的单转染均以pCre-IRES-EGFP为阳性对照组,分别对诱导表达出的Cre重组酶基因进行RT-PCR鉴定。
结果显示,经酶切和测序鉴定,成功构建了P1和P2载体。采用脂质体LP2000将P1与pTet-On质粒共转染293T细胞后,不加Dox时,没有或者很少表达绿色荧光;阳性对照组表达绿色荧光。而加入Dox后,P1与pTet-On质粒共转染的细胞诱导表达出绿色荧光;转染48小时后,分别提取其RNA,经RT-PCR检测表明,诱导表达出绿色荧光的细胞,均能检测出Cre基因的表达;反之,则检测不出Cre基因的表达,证实了绿色荧光和Cre基因的表达是一致的。
采用LP2000将P2转染293T细胞后,不加Dox时,没有绿色荧光的表达,阳性对照组表达绿色荧光,而加入Dox后,P2的转染能诱导表达绿色荧光;转染48小时后分别提取其RNA进行RT-PCR检测,诱导表达出绿色荧光时均能从mRNA水平上检测出Cre基因的表达;反之,则检测不出Cre基因的表达。
综上所述,得出以下结论:
本研究成功构建并鉴定了诱导性表达Cre重组酶的载体(P1和P2)。P1和pTet-On质粒的共转染能在293T细胞中诱导表达绿色荧光,并能从mRNA水平上检测出Cre基因的表达。P2的转染也能在293T细胞中诱导表达绿色荧光,并能从mRNA水平上检测出Cre基因的表达。实验结果证明,本研究所构建的诱导性表达Cre的载体P1和P2均能以时间特异性的方式诱导表达Cre重组酶基因,从细胞水平上证实了P1和P2载体的基因元件在体外均能发挥正常的功能,可以用于制备诱导性表达Cre重组酶的转基因小鼠。而且,使用P2载体仅需制备一种转基因小鼠就能满足研究的需要。
Conditional gene knockout mediated by Cre/loxP system is one of the most important technologies for the study of gene function.It circumvented some limitations of conventional knockout technology.On its basis , the inducible conditional gene knockout technology which combinates Tet gene expression system with Cre / loxP system can be used to study the function of genes more precisely.This technology requires two types of genetically engineered mice,one type is transgenic mice which can express Cre recombinase in the inducible manner,the other is gene targeting mice with loxP sites on both sides of targeting gene fragments.By the cross breeding of these two mouse lines, the inducible time-specific gene knockout can be achieved.
In this study two eukaryotic expression vectors were constructed and identified.They can inducibly express Cre recombinase. And the transcription activity of the vectors was also studied. This study lays the foundation for the generation of the mice inducibly expressing Cre and the conditional ADAM10 gene knockout mice, which will be helpful to understand the function of ADAM 10 gene.
By using the molecular biology technology, pTRE vectors of the Tet-On Gene Expression System were linearized and ligated to a 2.7kb fragments from the plasmid pCre-IRES-EGFP, as a result, the recombinant plasmid pTRE-Cre-EGFP (P1)was constructed.Then P1 and the pTet-On plasmid which was also from the Tet-On Gene Expression system were cotransfected into 293T cells with or without Dox, we observed whether or not there would be the green fluorescence and the expression of Cre recombinase. Phcmv-Cre-EGFP DNA fragments from the P1 vectors were ligated to Pcmv-rtTA fragments from the pTet-On vectors to form the vector P2. Then P2 vectors was transfected into 293T cells with or without Dox, we also observed green fluorescence and the expression of Cre recombinase, respectively.Plasmid pCre-IRES-EGFP was used as a positive control,either in the cotransfection of P1 and pTet-On or in the transfection of P2. And the inducible expression of Cre recombinase was identified by RT-PCR.
Result: Restriction endonuclease digestion and sequencing showed that vector P1 and P2 were successfully constructed.After P1 and pTet-On plasmid were cotransfected into 293T cells mediated by LP2000, without the existence of Dox,there was little or no expression of green fluorescence,while the positive control group expressed green fluorescence. after dox was added, the cotransfection of P1 and the pTet-On plasmid could induce the expression of green fluorescence. 48 hours after the transfection, RNAs were extracted for RT-PCR detection, respectively. The expression of Cre gene can be detected at mRNA levels, so long as the green fluorescence was induced.Otherwise, there was no expression of Cre gene.After P2 was transfected into 293T cells mediated by LP2000, without the Dox,there was little or no expression of green fluorescence.Only the positive control group expressed green fluorescence. But when dox was added, the transfection of P2 could induce the expression of green fluorescence. 48 hours after the transfection, RNAs were extracted for RT-PCR detection. When green fluorescence was induced, the expression of Cre gene can be detected at the mRNA level.Or there was no expression of Cre gene.
In conclusion, we successfully constructed and identified vectors that can inducibly express Cre recombinase (P1 and P2). The cotransfection of P1 and the pTet-On plasmid can induce the expression of green fluorescence in 293T cells.And the transfection of P2 can also induce the expression of green fluorescence in 293T cells.And the expression of Cre gene can be detected at the mRNA level. The results prove that the vector P1 and P2 we constructed induce the expression of Cre recombinase gene in time-specific manner, confirming that the gene elements in the vector P1 and P2 play a normal role in the cells. Both vector P1 and P2 can be used to prepare the Cre transgenic mouse. Moreover, we only prepare a kind of transgenic mice with the vector P2, which will satisfy the requirements for our research.
引文
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[16]Aiba A,Nakao H. Conditional mutant mice using tetracycline-Controlled gene expression system in the brain. Neurosci.Res, 2007,58:113-117.
2. Gossen M, Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA ,1992,89:5547–5551.
3. Gossen M, Freundlieb S, Bender G, et al. Transcriptional activation by tetracyclines in mammalian cells. Science,1995,268:1766–1769.
4. Gennady Ermak, Vincenzo J.Cancasci, Kelvin J.A.Davies, et al.Cytotoxic effect of doxycycline and its implications for tet-on gene expression systems. Analytical Biochemistry, 2003, 318: 152–154.
5.郑敬民,李坚等。条件性基因打靶及其研究进展。国外医学遗传学分册, 2001 ,24:236-240.
6. Zhong-Min GUO, Kang XU, Ying YUE, et al. Temporal Control of Cre Recombinase-mediated in Vitro DNA Recombination by Tet-on Gene Expression System. Acta Biochimica et Biophysica Sinica, 2005, 37: 133–138.
7. Lewandoski M. Conditional control of gene expression in the mouse. Nat Rev Genet, 2001, 2: 743–755.
8. Ryding AD, Sharp MG, Mullins JJ. Conditional transgenic technologies. J Endocrinol, 2001, 171: 1–14
9. van der Weyden L, Adams DJ, Bradley A. Tools for targeted manipulation of the mouse genome. Physiol Genomics, 2002, 11: 133–164.
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