發(fā)布時(shí)間：2018-05-16 11:32

  本文選題：條件性基因敲除 + 載體構(gòu)建　； 參考：《福建醫(yī)科大學(xué)》2008年碩士論文

【摘要】： Cre-loxP系統(tǒng)介導(dǎo)的條件性基因敲除(conditional gene knockout)是基因功能研究最重要的技術(shù)之一,它克服了傳統(tǒng)基因敲除導(dǎo)致小鼠胚胎死亡等諸多弊端。將該技術(shù)應(yīng)用于神經(jīng)系統(tǒng),可使基因敲除僅發(fā)生于大腦的某個(gè)亞區(qū)或發(fā)育過(guò)程的某個(gè)階段,以便對(duì)小鼠大腦基因功能進(jìn)行精細(xì)研究。對(duì)于神經(jīng)系統(tǒng)條件性基因敲除而言,選擇一個(gè)大腦亞區(qū)和時(shí)間特異性啟動(dòng)子指導(dǎo)Cre的表達(dá)是至關(guān)重要的。許多研究表明,CaMKⅡα基因的表達(dá)具有較強(qiáng)的時(shí)間和空間特異性[1-4],因此,符合制備前腦神經(jīng)細(xì)胞特異性表達(dá)Cre轉(zhuǎn)基因小鼠的要求。 本研究通過(guò)克隆和鑒定CaMKⅡα基因5’端不同調(diào)節(jié)區(qū)的序列,構(gòu)建了兩個(gè)神經(jīng)細(xì)胞特異性表達(dá)Cre的真核表達(dá)載體,并對(duì)這兩個(gè)載體的轉(zhuǎn)錄活性進(jìn)行了初步研究。為建立前腦神經(jīng)細(xì)胞特異性表達(dá)Cre的轉(zhuǎn)基因小鼠及進(jìn)一步制備ADAM10基因條件性敲除的小鼠奠定了基礎(chǔ),有助于開(kāi)展阿爾茨海默病發(fā)病機(jī)制的研究。 運(yùn)用PCR技術(shù),從C57BL/6J小鼠基因組中分步擴(kuò)增了CaMKⅡα基因5’端兩段調(diào)控序列,長(zhǎng)度分別為5.26kb和8.1kb。經(jīng)部分測(cè)序鑒定后,將片段分別連接于pCre-EGFP載體的Cre基因上游,構(gòu)建了神經(jīng)細(xì)胞特異性表達(dá)Cre重組酶的表達(dá)載體pCaMKⅡα-Cre-EGFPⅠ(pCCEⅠ)和pCaMKⅡα-Cre-EGFPⅡ(pCCEⅡ)。取C57BL/6J新生鼠與4周齡小鼠的端腦與海馬進(jìn)行神經(jīng)元的原代培養(yǎng)。分別以pCCEⅠ和pCCEⅡ?yàn)閷?shí)驗(yàn)組,pCre-EGFP為陽(yáng)性對(duì)照組,采用脂質(zhì)體2000(LP2000)轉(zhuǎn)染神經(jīng)細(xì)胞、小鼠神經(jīng)瘤與大鼠神經(jīng)膠質(zhì)瘤之融合細(xì)胞(NG108-15)和人乳腺癌細(xì)胞(ZR-75-30),研究pCCEⅠ和pCCEⅡ的表達(dá)活性及其組織特異性。 結(jié)果顯示,克隆的兩個(gè)CaMKⅡα基因5’端側(cè)翼區(qū)片段的酶切圖譜與公布的C57BL/6J小鼠相應(yīng)序列的酶切位點(diǎn)相一致,測(cè)序結(jié)果表明,所測(cè)序列與數(shù)據(jù)庫(kù)公布的C57BL/6J小鼠相應(yīng)序列基本相同,僅在-940bp(A→C)和-1.4kb(A→G)兩處存在差異。其中,文獻(xiàn)報(bào)道的富含順式調(diào)控元件的-900bp內(nèi)序列[5]與公布的C57BL/6J小鼠序列完全一致。5.26kb與8.1kb這兩段CaMKⅡα基因啟動(dòng)子均正確地連接于Cre基因的5’端,成功構(gòu)建了目標(biāo)載體。新生鼠神經(jīng)細(xì)胞的原代培養(yǎng)表明,細(xì)胞數(shù)量多(35mm培養(yǎng)皿中細(xì)胞密度"g106/cm3),細(xì)胞形態(tài)好。4周齡鼠神經(jīng)細(xì)胞的原代培養(yǎng)細(xì)胞形態(tài)較典型,但細(xì)胞數(shù)量很少(35mm培養(yǎng)皿中細(xì)胞不超過(guò)300個(gè))。用LP2000轉(zhuǎn)染后,在NG108-15細(xì)胞中,pCCEⅠ組能表達(dá)綠色熒光(表達(dá)率低),pCCEⅡ組不表達(dá)綠色熒光,陽(yáng)性對(duì)照組表達(dá)綠色熒光;在ZR-75-30細(xì)胞中,pCCEⅠ組和pCCEⅡ組都不表達(dá)綠色熒光,陽(yáng)性對(duì)照組表達(dá)綠色熒光;在新生鼠神經(jīng)細(xì)胞中,pCCEⅠ組和pCCEⅡ組都不表達(dá)綠色熒光,陽(yáng)性對(duì)照組表達(dá)綠色熒光(轉(zhuǎn)染效率低);在4周齡鼠神經(jīng)細(xì)胞中,由于細(xì)胞數(shù)量少,培養(yǎng)困難,經(jīng)LP2000處理后即發(fā)生死亡,故實(shí)驗(yàn)組與陽(yáng)性對(duì)照組均未表達(dá)綠色熒光。 綜上所述,得出以下結(jié)論: 本研究成功構(gòu)建并鑒定了神經(jīng)細(xì)胞特異性Cre表達(dá)載體(pCCEⅠ和pCCEⅡ)。成功培養(yǎng)了新生鼠與4周齡鼠的皮質(zhì)及海馬神經(jīng)細(xì)胞。pCCEⅠ能在神經(jīng)細(xì)胞瘤-神經(jīng)膠質(zhì)瘤細(xì)胞NG108-15中表達(dá)綠色熒光,說(shuō)明pCCEⅠ的啟動(dòng)子CaMKIIα基因5.26kb片段具有啟動(dòng)轉(zhuǎn)錄的活性;pCCEⅠ和pCCEⅡ在人乳腺癌細(xì)胞ZR-75-30中不表達(dá),支持了其表達(dá)的神經(jīng)細(xì)胞特異性;pCCEⅠ和pCCEⅡ在新生鼠神經(jīng)細(xì)胞中不表達(dá),一定程度上支持了其表達(dá)的時(shí)間特異性。pCCEⅡ中包含的CaMKIIα基因幾乎所有調(diào)控區(qū)的8.1kb片段的轉(zhuǎn)錄活性尚有待體內(nèi)實(shí)驗(yàn)的證實(shí)。實(shí)驗(yàn)結(jié)果證明本研究所構(gòu)建的神經(jīng)細(xì)胞特異性表達(dá)Cre的轉(zhuǎn)基因載體pCCEⅠ具有轉(zhuǎn)錄活性,且支持其表達(dá)的時(shí)空特異性。理論上證明了建立前腦神經(jīng)細(xì)胞特異性表達(dá)Cre重組酶的轉(zhuǎn)基因小鼠的可行性。
[Abstract]:The Cre-loxP system mediated conditional gene knockout is one of the most important techniques for gene function research. It overcomes the disadvantages of the traditional gene knockout, which leads to the death of the mouse embryo, and the application of this technique to the nervous system can only be used in some subregion of the brain or a certain stage of development. In order to make a fine study of gene function in the brain of mice, it is essential to select a subregion of the brain and a time specific promoter to guide the expression of Cre for the conditional gene knockout of the nervous system. Many studies have shown that the expression of the CaMK II alpha gene has a strong time and spatial specific [1-4], thus conforming to the preparation of the gene. Forebrain neurons specifically expressed Cre transgenic mice.
In this study, by cloning and identifying the sequence of the different regulation areas of the 5 'end of the CaMK II - a gene, two eukaryotic expression vectors expressing the specific expression of Cre were constructed, and the transcriptional activity of these two vectors was preliminarily studied. In order to establish the transgenic mice with the specific expression of Cre in the forebrain nerve cells and to further prepare the ADAM10 gene strip Knockout mice laid the foundation for the study of the pathogenesis of Alzheimer's disease.
PCR technique was used to amplify the two segments of the 5 'end of the CaMK II alpha gene from the C57BL/6J mouse genome, and the length was 5.26kb and 8.1kb. were identified by partial sequencing. The fragments were connected to the Cre gene of the pCre-EGFP vector respectively, and the expression vector of the Cre recombinant enzyme was constructed, and the expression vector of pCaMK II alpha -Cre-EGFP I was constructed. (pCCE I) and pCaMK II alpha -Cre-EGFP II (pCCE II). Primary culture of neurons in the end brain and hippocampus of 4 weeks old mice and 4 weeks old mice was cultured. PCCE I and pCCE II were used as experimental group, pCre-EGFP as positive control group, liposome 2000 (LP2000) transfected nerve cells, mouse neuroma and rat glioma fusion cells (NG). 108-15) and human breast cancer cells (ZR-75-30), to study the expression activity and tissue specificity of pCCE I and pCCE II.
The results showed that the cloned two CaMK II alpha gene 5 'end flanking region fragment was consistent with the corresponding sequence of the published C57BL/6J mice. The sequencing results showed that the sequence was basically the same as the corresponding sequence of C57BL/6J mice published by the database, and there were only two differences in -940bp (A to C) and -1.4kb (A to G). The -900bp internal sequence [5] rich in cis regulation elements and the published C57BL/6J mouse sequence fully conformed to the sequence of.5.26kb and 8.1kb, the two segment CaMK II alpha gene promoter correctly connected to the 5 'of the Cre gene and successfully constructed the target carrier. The primary culture of the newborn rat neural cells showed that the number of cells was much (the cell in 35mm culture dish). Density "g106/cm3"), the cell morphology of.4 weeks old rat neural cells is more typical, but the number of cells is little (no more than 300 cells in 35mm culture dish). After LP2000 transfection, in NG108-15 cells, pCCE I group can express green fluorescence (low expression rate), pCCE II group does not express green fluorescence, and the positive control group is green. In ZR-75-30 cells, both pCCE I group and pCCE II group did not express green fluorescence, and the positive control group expressed green fluorescence. In the newborn rat neural cells, both pCCE I group and pCCE II group did not express green fluorescence, and the positive control group expressed green fluorescence (low transfection efficiency). In the 4 week old rat neural cells, the number of cells was less and the culture was poor. It is difficult to die after LP2000 treatment. Therefore, green fluorescence was not expressed in both the experimental group and the positive control group.
To sum up, the following conclusions are drawn:
This study successfully constructed and identified the neural cell specific Cre expression vector (pCCE I and pCCE II). The cortical and hippocampal neurons of the 4 week old rats were successfully cultured and.PCCE I could express the green fluorescence in the neuroglioma cell NG108-15 cell NG108-15, indicating that the CaMKII alpha gene 5.26kb fragment of the promoter of pCCE I has a starting point. The activity of transcriptional transcription; pCCE I and pCCE II are not expressed in human breast cancer cell ZR-75-30, supporting their expressed Neurocellular specificity; pCCE I and pCCE II are not expressed in neonatal rat neural cells, and to some extent support the 8.1kb fragment of the CaMKII a gene contained in the time specific.PCCE II expressed in the expression of the 8.1kb fragment of the CaMKII a gene. The transcriptional activity has yet to be confirmed in the experiment in vivo. The experimental results show that the transgenic vector pCCE I, which is specifically expressed by Cre, has the transcriptional activity and supports the spatio-temporal specificity of its expression. The feasibility of establishing transgenic mice with the specific expression of Cre recombinant enzyme in the forebrain neural cells is theoretically proved.

【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R346

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 林春陽(yáng);陳亮;羅進(jìn)勇;鄧忠良;;雙表達(dá)骨形態(tài)發(fā)生蛋白2、9重組腺病毒載體的構(gòu)建和表達(dá)[J];中國(guó)微生態(tài)學(xué)雜志;2011年07期

2 郭冉;劉潔婷;劉海峰;張春軍;初彥輝;;TGF-β1原核表達(dá)載體的構(gòu)建[J];牡丹江醫(yī)學(xué)院學(xué)報(bào);2011年04期

3 胡國(guó)棟;陳英華;佟萬(wàn)成;程遠(yuǎn)雄;張琳;張磊;蔡紹曦;;血管內(nèi)皮細(xì)胞特異性敲除cdc42基因的雜合子小鼠與非基因敲除小鼠在急性肺損傷肺組織病理改變以及肺微血管通透性變化的比較[J];南方醫(yī)科大學(xué)學(xué)報(bào);2011年06期

4 孔凡銘;張新偉;于津浦;魏楓;李慧;任秀寶;;iASPP真核表達(dá)載體的構(gòu)建及其對(duì)NIH3T3細(xì)胞增殖的影響[J];天津醫(yī)科大學(xué)學(xué)報(bào);2011年02期

5 徐蘭蘭;游莉;郭元元;鄒正渝;黎玉葉;孫雙雙;羅進(jìn)勇;周蘭;;人S100 A9重組腺病毒載體的構(gòu)建和鑒定[J];重慶醫(yī)學(xué);2011年22期

6 李田田;劉明;魏鑒騰;吳寧;王翠翠;林秀坤;;RSK4基因表達(dá)及其對(duì)U87細(xì)胞增殖的影響[J];中國(guó)神經(jīng)腫瘤雜志;2011年02期

7 朱偉;曹以誠(chéng);楊磊;;整合siRNA技術(shù)的結(jié)核核酸疫苗的AAV載體構(gòu)建與濃縮純化[J];現(xiàn)代食品科技;2011年07期

8 柯大智;陳慶偉;李桂瓊;鄧瑋;莫顯剛;;insig-1基因siRNA干擾質(zhì)粒的構(gòu)建及對(duì)炎癥狀態(tài)下細(xì)胞膽固醇代謝的影響[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2011年14期

9 孫宇;羅佳;馬玉媛;章金剛;;豬APOBEC3F基因克隆及真核表達(dá)載體的構(gòu)建與鑒定[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2011年18期

10 向瓊;唐惠芳;殷杰;虞佳;楊曉燕;朱炳陽(yáng);雷小勇;;MicroRNA-98對(duì)A549/DDP細(xì)胞順鉑耐藥性的影響[J];中南醫(yī)學(xué)科學(xué)雜志;2011年03期

相關(guān)會(huì)議論文 前10條

1 溫丹萍;王際平;王慧君;張進(jìn);梁菲;楊曉;費(fèi)儉;王鑄鋼;馬端;;組織因子途徑抑制物-1血管條件性基因敲除小鼠表型和功能分析[A];中國(guó)的遺傳學(xué)研究——遺傳學(xué)進(jìn)步推動(dòng)中國(guó)西部經(jīng)濟(jì)與社會(huì)發(fā)展——2011年中國(guó)遺傳學(xué)會(huì)大會(huì)論文摘要匯編[C];2011年

2 馬君;章金剛;向華;李雪梅;史秋梅;宣華;;鵝細(xì)小病毒主要結(jié)構(gòu)蛋白基因的克隆、測(cè)序及載體的構(gòu)建[A];中國(guó)畜牧獸醫(yī)學(xué)會(huì)禽病學(xué)會(huì)分會(huì)第十次學(xué)術(shù)研討會(huì)論文集[C];2000年

3 師偉;裴雪濤;;HOXB4基因的表達(dá)譜初步研究與HOXB4基因的克隆及相關(guān)載體構(gòu)建[A];第九屆全國(guó)實(shí)驗(yàn)血液學(xué)會(huì)議論文摘要匯編[C];2003年

4 徐建華;何敏;黃憲章;柯培鋒;林蓮英;黎美賢;莊俊華;;Survivin shRNA載體構(gòu)建及靶向下調(diào)PC3 mRNA表達(dá)實(shí)驗(yàn)研究[A];中華醫(yī)學(xué)會(huì)第九次全國(guó)檢驗(yàn)醫(yī)學(xué)學(xué)術(shù)會(huì)議暨中國(guó)醫(yī)院協(xié)會(huì)臨床檢驗(yàn)管理專(zhuān)業(yè)委員會(huì)第六屆全國(guó)臨床檢驗(yàn)實(shí)驗(yàn)室管理學(xué)術(shù)會(huì)議論文匯編[C];2011年

5 劉文文;趙永波;;大鼠GDNF真核表達(dá)載體構(gòu)建[A];中華醫(yī)學(xué)會(huì)第七次全國(guó)神經(jīng)病學(xué)學(xué)術(shù)會(huì)議論文匯編[C];2004年

6 李克;王琳;成軍;張玲霞;陸蔭英;李莉;;丙型肝炎病毒核心蛋白基因的表達(dá)載體構(gòu)建及在酵母中的表達(dá)[A];中華醫(yī)學(xué)會(huì)第七次全國(guó)感染病學(xué)術(shù)會(huì)議論文匯編[C];2001年

7 李臻;金寧一;金洪濤;鄭敏;付延軍;辛蘇文;;新型雞痘病毒重組轉(zhuǎn)移載體的構(gòu)建[A];中國(guó)畜牧獸醫(yī)學(xué)會(huì)家畜傳染病學(xué)分會(huì)第六屆理事會(huì)第二次會(huì)議暨教學(xué)專(zhuān)業(yè)委員會(huì)第六屆代表大會(huì)論文集[C];2006年

8 肖仕初;夏照帆;朱世輝;郇京寧;楊s，

本文編號(hào)：1896658