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研究生:蔡夙美
研究生(外文):Su-mei Tsai
論文名稱:建立在乳腺表達TV-A受體之基因轉殖小鼠
論文名稱(外文):Generation of transgenic mouse model expressing TV-A receptor in the mammary gland
指導教授:曾英傑曾英傑引用關係
學位類別:碩士
校院名稱:慈濟大學
系所名稱:人類遺傳學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
畢業學年度:91
語文別:英文
論文頁數:55
中文關鍵詞:禽類反轉錄病毒載體轉殖小鼠乳腺TV-A受體
外文關鍵詞:RCAS vectortransgenic micemammary glandTV-A receptor
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二十世紀的分子生物學主要的研究方向為將一複雜的生物現象區分為數個不同的生化途徑並分別探討其機制,而現今的分子生物學家則試圖將這些個別的生化途徑整合起來,以了解這些複雜的生物現象並解讀其所代表的意義。嘗試達到此目的的諸多方法中,基因剔除小鼠以及基因轉殖小鼠的建立在研究一特定基因在活體內所具有的生理功能中,扮演了舉足輕重的角色。基因剔除小鼠以及基因轉殖小鼠已被運用於許多方面的研究,例如致癌的機轉以及器官的發育。腫瘤或是器官的形成都是由多個基因間相互調控所導致的結果,然而以傳統的基因剔除或是基因轉殖法所做出來的小鼠模式一次只能改變一種基因的表達量,若要在小鼠模式上研究多個基因間的相互影響,則除了分別建立帶有不同外源基因的小鼠株外,還要透過繁複的交配過程才能獲得帶有多基因的轉殖小鼠。為了解決此一問題,Harold Varmous等人在1994年建立了一套應用在小鼠模式上的外源基因運送系統。此系統是將禽類反轉錄病毒ASLV-A (avian sarcoma and leukosis virus)的受體TV-A以基因轉殖的方式導入小鼠體內,再以具有組織特異性的促進子使特定小鼠細胞表達TV-A受體;另外為了使ASLV-A能攜帶外源基因,Steve Hughes等人在1987年將ASLV-A加以改造並以DNA載體的方式呈現,稱為RCAS載體(replication-competent ASLV-A with splice acceptor)。在進行外源基因功能的探討時,可將外源基因置入RCAS載體並轉染至禽類細胞並將帶有外源基因的改造病毒大量製造出來後,即可透過轉殖小鼠特定細胞表面的TV-A受體以病毒感染的方式將外源基因運送至細胞內。為了將RCAS-TV-A應用在乳腺,我們選擇具有乳腺上皮細胞特異性的乳清酸性蛋白促進子(whey acidic protein promoter, WAP promoter)來調控TV-A受體表達。藉由單細胞時期之鼠胚顯微注射法成功地建立了六隻帶有tv-a轉殖基因之轉殖小鼠,實驗證明轉殖基因在這些轉殖小鼠中有四隻能穩定地傳遞到牠們的子代當中。分析了轉殖小鼠乳腺的RNA與蛋白質後得知:在WAP啟動子的調控之下,轉殖基因於小鼠株-2、小鼠株-5與小鼠株-6泌乳期母鼠的乳腺中均能被順利地表達出來。未來將以帶有綠色螢光蛋白報導基因的病毒透過TV-A受體感染乳腺上皮細胞,經過冷凍切片在螢光顯微鏡下觀察綠色螢光蛋白有無表達,以確認小鼠株乳腺上皮細胞所表達的TVA受體具有能使帶有外源基因的ASLV-A感染的能力後,就可以將各種外源基因如抑癌基因透過RCAS-TVA系統送至乳腺並進一步研究這些外源基因對乳腺以及乳癌形成的影響。
The twentieth-century reductionist approach to molecular biology dissected complex biological phenomena into distinct biochemical pathways. Today, we’re challenged by these distinct biochemical pathways in an attempt to recapitulate all the complex biological settings. Gene knockout and transgenic mice have yielded significant insights into the contribution of specific genes to developmental process or oncogensis in vivo. The tumorigenesis and organogenesis are thought to be the result of interactions between multiple genes. However, detecting cooperation between alterations generated by these conventional techniques requires complex crossing between different transgenic mouse lines. To circumvent this problem, the lab of Harold Varmus developed a flexible gene delivery system. This system was based on modification of the subtype A of avian sarcoma and leukosis virus (ASLV-A) into a proviral vector to form replication-competent ASLV-A with splice acceptor (RCAS) and expression of TV-A receptor for the modified ASLV-A under the control of various mammalian promoters in transgenic mice, thus rendering mice susceptible to infection with ASLV-A. In order to develop a mammary-specific gene delivery system, We adapted the RCAS-TV-A system and generated several WAP-TV-A transgenic mouse lines. To generate WAP-TV-A transgenic mice, I microinjected the WAP-TV-A construct into pronuclei of one-cell stage mouse embryos. To detect the genomic integration of the transgene tv-a, I extracted DNA from tails of mice at 2-3 weeks of age and analyzed DNA by PCR and Southern blot. I thus obtained six founder mice and generated four individual mouse lines. I further confirmed the expression signals of the TV-A receptor in the mammary gland of lactating transgenic mice by RT-PCR and Northern blot analysis. Three transgenic lines expressing tv-a mRNA specifically in the mammary gland were detected. I also detected the TV-A protein expression by Western blot analysis, and the result was consistent with the Northern blot analysis. After the biological function of these WAP-TV-A transgenic mice being tested, a number of genetic lesions can be introduced simultaneously or sequentially into individual cells in a TV-A transgenic mouse, thus allowing for the study of numerous combinations of genetic alterations in mammary gland.
Content 1. INTRODUCTION 7 1.1. TRANSFER OF FOREIGN GENES INTO MAMMALIAN CELLS 7 1.1.1. DNA transfer strategies 7 1.2. PRINCIPLE METHODS TO GENERATE TRANSGENIC MOUSE MODELS 8 1.2.1. Pronuclear injection 9 1.2.2. Blastocyst injection 9 1.2.3. Virus infection: retroviral vectors 9 1.2.4. Disadvantages of current methodologies 10 1.3. THE RCAS-TV-A SYSTEM 11 1.3.1. The TV-A receptor 11 1.3.2. The ASLVs 11 1.3.3. The RCAS vector 12 1.3.4. Advantages of RCAS-TV-A system for gene transfer in the mammalian system 13 1.4. ADVANTAGES OF THE MAMMARY GLAND AS A MODEL SYSTEM 13 1.5. WHEY ACIDIC PROTEIN (WAP) GENE PROMOTER 14 1.5.1. Structure of whey acidic protein promoter (WAPpr) 14 1.5.2. Regulation of WAP promoter activity by hormones 14 1.5.3. Regulation of the WAP promoter by cell-cell interactions 15 1.5.4. Applications of driving transgene expression in the mammary gland by the WAP promoter 15 1.6. THE PURPOSE OF THIS THESIS 16 2. MATERIALS AND METHODS 17 2.1. MATERIALS 17 2.1.1. Animals 17 2.1.2. Microinjection and Embryo Transfer 17 2.1.3. DNA Analysis 18 2.1.4. RNA Analysis 20 2.1.5. Protein Analysis 21 2.2. METHODS 22 2.2.1. Microinjection and Embryo Transfer 22 2.2.2. DNA Analysis 25 2.2.3. RNA Analysis 26 2.2.4. Protein Analysis 28 3. RESULTS 30 3.1. ESTABLISHMENT OF TRANSGENIC MICE CARRYING WAP-TV-A DNA 30 3.1.1. Preparation of WAP-TV-A construct 30 3.1.2. Founder mice and their transgenic progeny screened by PCR and Southern blot analysis 30 3.2. TRANSGENE WAP-TV-A EXPRESSION IN THE MAMMARY GLAND 31 3.2.1 Detection of TV-A RNA expression in the mammary gland by RT-PCR and Northern blot analysis 31 3.2.2 Western blot analysis 32 3.3. SIDE-EFFECT DETERMINATION OF TV-A PROTEIN EXPRESSION IN MOUSE MAMMARY GLAND 32 4. DISCUSSION 33 5. REFERENCES 37 Chart List TABLE1. MAMMALIAN EXPRESSION SYSTEMS 42 FIG.1. PROVIRAL VECTOR: RCAS 43 FIG.2. SCHEMATIC REPRESENTATION OF THE WAP PROMOTER 44 FIG.3. STEPS OF WAP-TV-A CONSTRUCTION 45 FIG.4. SCHEMATIC REPRESENTATION OF THE PRIMERS USED IN PCR DETECTION, SOUTHERN BLOT AND NORTHERN BLOT PROBE SYNTHESIS 46 5A: ILLUSTRATION OF RESTRICTION ENZYMES USED TO CHECK WAP-TV-A CONSTRUCTION 47 5B: RESTRICTION ENZYME DIGESTION AND ELECTROPHORESIS. 47 FIG.6 FOUNDER MICE SCREENED BY PCR ANALYSIS 48 FIG.7. FOUNDER MICE SCREENED BY SOUTHERN BLOT ANALYSIS 49 FIG.8. F1 TRANSGENIC MICE SCREENED BY PCR ANALYSIS 50 TABLE 2. SUMMARY OF WAP-TV-A TRANSGENESIS 51 FIG.9. TV-A RNA EXPRESSION OF TRANSGENIC MAMMARY GLAND WAS DETECTED BY RT-PCR WITH TV-A3/ TV-A4 PRIMER PAIRS 52 FIG.10. TV-A RNA EXPRESSION OF THE TRANSGENIC MAMMARY GLAND WAS DETECTED BY NORTHERN BLOT ANALYSIS 53 FIG. 11. TV-A RNA EXPRESSION SPECIFICITY WAS DETERMINED BY RT-PCR ANALYSIS 54 FIG. 12. TV-A PROTEIN EXPRESSION OF TRANSGENIC MAMMARY GLAND DETECTED BY WESTERN BLOT ANALYSIS. 55
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