Expression Analysis of Goat β-Casein/Human Serum Albumin Fusion Gene in Transgenic Mice

以本實驗室所選殖之山羊β-酪蛋白基因pGB5-1為模版，構築高表現能力之啟動子（promoter）序列，設計DNA引子，以β-酪蛋白5’端調節序列至exon 2為目標，利用PCR增殖出含exon 2之5’端β-酪蛋白基因調節序列。將其所增殖出之片段選殖入載體內，形成新質體pGB528（2.8 kb），pGB538（3.8 kb），pGB552（5.2 kb）及pGB565（6.5 kb）。經限制酶切割與南方吸漬分析，證實其序列之正確性，做為後續基因轉殖研究所需。 酪蛋白基因之表現具有組織與階段專一性，可在泌乳相關內泌素及細胞外基質（extracellular matrix）之刺激下，在乳腺上皮細胞中表現。選殖5’端調節序列並包含exon 1、intron 1及exon 2的山羊β-酪蛋白基因（-4044至 +2123），與綠色螢光蛋白（green fluorescent protein, GFP）報導基因構築成新載體pGB562/GFP。將之以脂質體（liposome）轉染至小鼠乳腺上皮細胞株NMuMG中，觀察融合基因表現能力。pGB562/GFP轉染之NMuMG細胞經過24，48及72 h培養後，其GFP之表現明顯高出控制組分別有25，55及42倍之多。另以電穿孔法（electroporation）轉形至離體培養之泌乳母鼠乳腺組織，觀察融合基因表現能力，以螢光顯微鏡或雷射共軛焦顯微鏡（laser scanning confocal microscope）觀察，結果顯示離體培養24 h之乳腺組織中出現明顯綠色螢光。由此證明所選殖之6.2 kb山羊β-酪蛋白基因5’端調節序列GB562具有優越的啟動基因表現能力，可供後續連結其他外源蛋白基因之用。 另一研究主題則是嘗試以體外受精（in vitro fertilization; IVF）技術與改變體外受精培養液成分，提高受精率，作為產製基因轉殖鼠所需受精卵之來源。試驗探討影響BALB/c小鼠IVF之因素，及改變培養液葡萄糖濃度，以提高其受精率之可行性。取BALB/cByJ 與FVB/NCrl 公小鼠精子，與母小鼠經超數排卵處理之卵子，進行體外受精，評估其受精率。實驗結果顯示，以FVB/NCrl公鼠為精子供應者，其受精率平均為69.6%；而BALB/cByJ公鼠為精子供應者，其受精率平均為12.1%。再者，培養液組成分中葡萄糖為提供精子能量的主要來源，故本研究探討改變培養液葡萄糖濃度對BALB/cByJ小鼠IVF之影響。試驗結果顯示，在TYH培養液含有0、2.7、5.5、11.1及22.2 mM葡萄糖組中，BALB/cByJ小鼠體外受精後發育至2-細胞期胚之比率分別為6.8、9.9、13.9、32.7及22.2%。故適度增加培養液中葡萄糖濃度，可增加BALB/cByJ精子IVF之效率。 應用所選殖的山羊β-酪蛋白基因5’端調節序列產製基因轉殖鼠。試驗以選殖自人類肝臟cDNA基因庫，涵蓋1830 bp完整胺基酸轉譯序列之人類血清白蛋白（human serum albumin, HSA）基因，與山羊β-酪蛋白基因5’端調節序列GB562連接構築成pGB562/HSA （12.5 kb）重組質體。以含-酪蛋白啟動子、HSA及SV40 polyadenylation signals之片段（9.6 kb）進行小鼠受精卵之顯微注射，以產製基因轉殖小鼠，並觀察轉殖之人類血清白蛋白基因在小鼠之表現。經由PCR及南方吸漬雜交(Southern blot)，證實3隻小鼠（2母1公）為基因轉殖小鼠。轉殖基因嵌入的套數在＃21，＃26族系超過10個套數，在＃31族系超過50個套數。收集母鼠乳汁進行西方吸漬雜交(Western blot)，確認基因轉殖小鼠乳汁中含有HSA之蛋白產物，其濃度介於0.05 ~ 0.4 mg/mL之間。基因轉殖小鼠乳腺之切片經免疫化學染色，可於乳腺泡內出現明顯HSA抗原反應。HSA基因轉殖鼠子代母鼠，取其乳腺組織，抽取RNA進行RT-PCR，結果確定基因可傳遞至子代並可轉錄HSA轉殖基因。The purpose of this study was to amplify DNA sequences by PCR to generate the regulatory sequence of goat β-casein gene for serial studies of gene expression in mammalian cells or transgenic animals. Several primers were designed for PCR to amplify DNA sequences from 5' end to exon 2 of goat β-casein gene according to the template plasmid pGB5-1. The 2.8, 3.8, 5.2 and 6.5 kb DNA fragments were obtained in the PCR products to form new vectors pGB528, pGB538, pGB552 and pGB565, respectively. In mammary epithelial cells, the stage and tissue-specific expression of the β-casein gene is under the control of lactogenic hormone prolactin, glucocorticoids and dependent on extracellular matrix components. The 5' regulative sequence and intron 1 of the goat β-casein gene from -4044 to +2123 bp, was cloned and fused with the reporter gene of green fluorescent protein (GFP) to create a plasmid termed pGB562/GFP. To detect GFP expression, pGB562/GFP was transfected in vitro via liposomes into the mammary epithelial cell line NMuMG. Cells could not express GFP unless the transfected NMuMG cells lined up to create functional alveoli. These functional cells were cultured with lactogenic hormones, including insulin, dexamethasone and prolactin, and were grown on a layer of the extracellular matrix Matrigel. Green fluorescent protein expression levels in NMuMG cells were 25-, 55- and 42-fold those in the control group at 24, 48, and 72 h after pGB562/GFP transfection respectively. In addition, pGB562/GFP was transfected ex vivo by electroporation into mammary gland fragments and cells were then cultured in vitro with a supplement of lactogenic hormones. Strong GFP expression localized in fragments of the mammary gland was observed 24 h after gene transfer. The novel strategy of ex vivo gene transfer into mammary tissue using GFP as a reporter gene to detect the function of a tissue-specific promoter is efficient and convenient. The data obtained herein reveal that the 5' regulative sequence and intron 1 including exon1 and 2 of the 6.2 kb goat β-casein gene can enhance the efficiency of transgene expression. Thus, the GB562 sequence may act as a good promoter and effectively elevate the production of exogenous protein in mammary glands. To provide fine quality fertilize eggs for generating high rate of transgenic mice, meanwhile, to probe into the genetic effect of mice in vitro fertilization (IVF) efficiency, we try to modify IVF technique and culture medium. BALB/c mice are widely used in genetic, tumour and immunological studies. Based on IVF, a routine technique for biomedical studies, it is worth to compare the effects of BALB/c mice with FVB/N on IVF efficiency. Four reciprocal IVF tests of BALB/cByJ and FVB/NCrl mice were performed. The results showed that the average fertility of IVF sponsored by FVB/NCrl spermatozoa was 69.6%, but only 12.1% was obtained from BALB/cByJ strain. Effect of glucose contained in the culture medium to the IVF efficiency of BALB/cByJ was also evaluated. The results showed that the fertility of BALB/cByJ spermatozoa incubated with 0, 2.7, 5.5, 11.1 and 22.2 mM of glucose in the TYH medium were 6.8, 9.9, 13.9, 32.7 and 22.2%, respectively. It is showed that IVF efficiency of BALB/cByJ spermatozoa could be improved depending on the increasing concentration of glucose in the IVF medium. The gene encoding human serum albumin (HSA) was cloned from human liver cDNA library by PCR. The HSA cDNA in size of 2,176 bp, including 1,830 bp of open reading frame, was cloned into the plasmid carried with the 5' flanking sequence of goat β-casein gene GB562 to form a tissue specific expression vector in mammary gland named pGB562/HSA (12.5 kb). A 9.6 kb DNA fragment in which the sequence is in order of goat β-casein gene regulatory sequence, HSA cDNA and SV40 polyadenylation signals was isolated from the pGB562/HSA, and used to microinject into the pronuclei of mouse fertilized eggs to produce transgenic mice. Three transgenic mice (2 female and 1 male) were identified by PCR and dot Southern blot analysis. The copy numbers of integrated transgene were more than 10 copies in line #21 and #26 as well as over 50 copies in line #31 of transgenic mice. HSA protein collected from the milk of lactating transgenic mice was confirmed by immuno-detection of Western and slot blot. The concentrations of HSA in the milk were from 0.05 to 0.4 mg/mL. An obvious antigen and antibody conjugate was seen at immunohistochemical stain of mammary gland tissue from lactating day 11 of HSA transgenic mice. The transmission of transgene and its expression was recognized according to the results of RT-PCR and sequences analyses of their progeny.圖 次 表 次 摘 要 緒 言 文獻檢討 一、乳中酪蛋白之性質 二、β-酪蛋白基因及其結構 三、影響乳蛋白基因表現之轉錄調節因子 四、人類血清白蛋白 第一章 山羊β-酪蛋白基因5’端調節序列之選殖與表現 第一節 山羊β-酪蛋白基因5’端調節序列之選殖 第二節 山羊β-酪蛋白/綠色螢光蛋白基因在乳腺細胞（in vitro） 及乳腺組織（ex vivo）之表現 第二章 培養液中葡萄糖濃度對小鼠體外受精之影響 第三章 山羊β-酪蛋白/人類血清白蛋白融合基因之基因轉殖小鼠產製 與基因表現測定 參考文獻 英文摘要 附