利用牛α-乳白蛋白啟動子驅動人類B型與C型肺臟表面張力相關蛋白之轉殖基因小鼠產製

Generation of Transgenic Mice Harboring the Human Pulmonary Surfactant-Associated Protein -B and -C Gene(s) Driven by Bovine α - Lactalbumin Promoter

李燕儒(Yen-Ju Lee)

指導教授 ： 吳信志

國立臺灣大學/生物資源暨農學院/動物科學技術學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.10197

摘要

摘要
本研究旨在嘗試構築由牛 α 乳白蛋白啟動子分別攜帶全長之B型與C型肺臟表面張力劑蛋白質 (SP-B 與SP-C) ，及成熟之SP-B與SP-C序列之轉殖基因片段，透過原核基因顯微操作，產製攜帶此基因之小鼠，待基因轉殖小鼠懷孕分娩後可由乳汁中獲得該兩種具生理醫療活性之蛋白質，經過純化，可獲得高量藥用蛋白。此可有效克服治療用肺臟表面張力劑多採用動物肺臟萃取含量稀少，純化不易且價格昂貴之缺憾。為達此目標，總共構築αLA-CNHIS-HSPB、αLA-CNHIS-HSPC、αLA-CNHIS-mHSPB 及 αLA-CNHIS—mHSPC四種轉殖基因，此四種轉殖基因分別為由牛 α乳白蛋白啟動子與山羊αs1酪蛋白之信息胜肽所調控之全長SPB、SPC與去除兩端序列之成熟SPB、SPC片段。於進行基因轉殖動物試驗之前，先行將此四段轉殖基因分別轉染小鼠乳腺上皮細胞。以PCR確定所轉染之乳腺上皮細胞的確帶有外源性基因後，將細胞分化處理，觀察各組細胞分化成為類腺泡構造時，其RNA與蛋白質表現情況。經RT-PCR與細胞免疫螢光染色後，初步證實四組轉殖基因得確可於分化之乳腺細胞中表現。爾後接著進行基因轉殖動物試驗。在αLA-CNHIS-mHSPB基因轉殖小鼠試驗中，共計移置309個業經注射該基因之小鼠胚，移至九隻代理孕母之兩側輸卵管內，共六隻代理孕母成功懷孕，並於懷孕期滿後陸續產下合計47隻仔小鼠。俟小鼠四周離乳後，採其尾部肌肉組織之基因組DNA。透過PCR、Slot-blot 與Southern-blot分析證實，有三隻係帶有αLA-CNHIS-mHSPB轉殖基因者。此等轉殖基因小鼠目前陸續發育至性成熟階段，俟其懷孕期滿分娩後，可進一步分析確認性腺被傳承之效率，並確認轉殖基因於轉殖基因母鼠乳腺中之表現情形。同樣地，αLA-CNHIS-mHSPC基因轉殖小鼠試驗中，總共移置249個業經顯微注射該基因之小鼠胚。將此249個小鼠胚分別移置九隻代理孕母兩側輸卵管中。待懷孕期滿，陸續產下24隻仔小鼠，除最早產出者遭母鼠攻擊死亡外，目前存活之仔小鼠約兩週齡左右，俟其離乳後，將進行進一步之分析。基因轉殖動物試驗仍持續進行中。未來期望可由基因轉殖小鼠乳汁中進一步探知兩組轉殖基因於動物體內之表現情形。

關鍵字

人類B型肺臟表面張力劑相關蛋白質 ； 人類C型肺臟表面張力劑相關蛋白質 ； 牛α-乳白蛋白啟動子 ； 基因轉殖小鼠

並列摘要

Generation and Analysis of Transgenic Mice Harboring the human pulmonary surfactant protein B and C Genes Driven by bovine α-Lactalbumin Promoter

Yen-Ju Lee

Abstract

This study establishes that the constructed bovine α- lactalbumin promoter could drive the full-length SP-B、S-C and mature SP-B、 S-C genes secreted in the milk, after purification, are potentially useful as pharmaceutical products.
To meet the purpose described above, a total of four transgenes, named αLACN-HIS-hSPB, αLACN-HIS-hSPC, αLACN-HIS-mhSPB and αLACN-HIS--mhSPC, were constructed for the use of subsequent studies related to generation of Tg animals. All these transgenes were designed to allow each of them containing either a full-length cDNA or a truncated mature cDNA sequences encoding a target protein of either SP-B or SP-C that was each driven by a promoter sequence from bovine α-lactalbumin (αLA) gene equipped with a signal peptide sequence from goat αs1-casein gene. Moreover, the expression potential of each transgene(s) had been confirmed by PCR, RT-PCR and Immunocytochemistry, based on the evidences found in those of mouse mammary gland cells post the gene(s) transfection, before they were subsequently subjected to microinjection into the pronucleus of mouse newly fertilized eggs.
Transfer of 309 mouse embryos, after microinjection of the αLACN-HIS-mhSPB transgene, into 9 recipient mice has resulted in six of them becoming pregnancy and a total of 47 full term pups have so far obtained from six of the pregnant surrogates and three of these 47 pups has been confirmed to be transgenic, according to results obtained from PCR and Southern-blot analyses against to genomic DNA extracted from tissue of the tail muscle. Similarly, transfer of 249 mouse embryos, after they been subjected to microinjection of the αLACN-HIS-mhSPC transgene, into 9 recipient mice has resulted in six of them becoming pregnancy and a total of 24 full term pups have so far obtained from four of the pregnant surrogates. The experiments for microinjection are still going.

並列關鍵字

Human pulmonary surfactant-associated protein B ； Human pulmonary surfactant-associated protein C ； Bovine α-Lactalbumin promoter ； transgenic mice

參考文獻

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