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

【摘要】： Cre-loxP系統(tǒng)介導的條件性基因敲除(conditional gene knockout)是基因功能研究最重要的技術之一,它克服了傳統(tǒng)基因敲除導致小鼠胚胎死亡等諸多弊端。將該技術應用于神經系統(tǒng),可使基因敲除僅發(fā)生于大腦的某個亞區(qū)或發(fā)育過程的某個階段,以便對小鼠大腦基因功能進行精細研究。對于神經系統(tǒng)條件性基因敲除而言,選擇一個大腦亞區(qū)和時間特異性啟動子指導Cre的表達是至關重要的。許多研究表明,CaMKⅡα基因的表達具有較強的時間和空間特異性[1-4],因此,符合制備前腦神經細胞特異性表達Cre轉基因小鼠的要求。 本研究通過克隆和鑒定CaMKⅡα基因5’端不同調節(jié)區(qū)的序列,構建了兩個神經細胞特異性表達Cre的真核表達載體,并對這兩個載體的轉錄活性進行了初步研究。為建立前腦神經細胞特異性表達Cre的轉基因小鼠及進一步制備ADAM10基因條件性敲除的小鼠奠定了基礎,有助于開展阿爾茨海默病發(fā)病機制的研究。 運用PCR技術,從C57BL/6J小鼠基因組中分步擴增了CaMKⅡα基因5’端兩段調控序列,長度分別為5.26kb和8.1kb。經部分測序鑒定后,將片段分別連接于pCre-EGFP載體的Cre基因上游,構建了神經細胞特異性表達Cre重組酶的表達載體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行神經元的原代培養(yǎng)。分別以pCCEⅠ和pCCEⅡ為實驗組,pCre-EGFP為陽性對照組,采用脂質體2000(LP2000)轉染神經細胞、小鼠神經瘤與大鼠神經膠質瘤之融合細胞(NG108-15)和人乳腺癌細胞(ZR-75-30),研究pCCEⅠ和pCCEⅡ的表達活性及其組織特異性。 結果顯示,克隆的兩個CaMKⅡα基因5’端側翼區(qū)片段的酶切圖譜與公布的C57BL/6J小鼠相應序列的酶切位點相一致,測序結果表明,所測序列與數據庫公布的C57BL/6J小鼠相應序列基本相同,僅在-940bp(A→C)和-1.4kb(A→G)兩處存在差異。其中,文獻報道的富含順式調控元件的-900bp內序列[5]與公布的C57BL/6J小鼠序列完全一致。5.26kb與8.1kb這兩段CaMKⅡα基因啟動子均正確地連接于Cre基因的5’端,成功構建了目標載體。新生鼠神經細胞的原代培養(yǎng)表明,細胞數量多(35mm培養(yǎng)皿中細胞密度"g106/cm3),細胞形態(tài)好。4周齡鼠神經細胞的原代培養(yǎng)細胞形態(tài)較典型,但細胞數量很少(35mm培養(yǎng)皿中細胞不超過300個)。用LP2000轉染后,在NG108-15細胞中,pCCEⅠ組能表達綠色熒光(表達率低),pCCEⅡ組不表達綠色熒光,陽性對照組表達綠色熒光;在ZR-75-30細胞中,pCCEⅠ組和pCCEⅡ組都不表達綠色熒光,陽性對照組表達綠色熒光;在新生鼠神經細胞中,pCCEⅠ組和pCCEⅡ組都不表達綠色熒光,陽性對照組表達綠色熒光(轉染效率低);在4周齡鼠神經細胞中,由于細胞數量少,培養(yǎng)困難,經LP2000處理后即發(fā)生死亡,故實驗組與陽性對照組均未表達綠色熒光。 綜上所述,得出以下結論: 本研究成功構建并鑒定了神經細胞特異性Cre表達載體(pCCEⅠ和pCCEⅡ)。成功培養(yǎng)了新生鼠與4周齡鼠的皮質及海馬神經細胞。pCCEⅠ能在神經細胞瘤-神經膠質瘤細胞NG108-15中表達綠色熒光,說明pCCEⅠ的啟動子CaMKIIα基因5.26kb片段具有啟動轉錄的活性;pCCEⅠ和pCCEⅡ在人乳腺癌細胞ZR-75-30中不表達,支持了其表達的神經細胞特異性;pCCEⅠ和pCCEⅡ在新生鼠神經細胞中不表達,一定程度上支持了其表達的時間特異性。pCCEⅡ中包含的CaMKIIα基因幾乎所有調控區(qū)的8.1kb片段的轉錄活性尚有待體內實驗的證實。實驗結果證明本研究所構建的神經細胞特異性表達Cre的轉基因載體pCCEⅠ具有轉錄活性,且支持其表達的時空特異性。理論上證明了建立前腦神經細胞特異性表達Cre重組酶的轉基因小鼠的可行性。
[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.

【學位授予單位】：福建醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R346

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