攜帶豬胰澱粉酶啟動子/纖維素分解酶、植酸分解酶轉基因小鼠及轉基因豬之產製與分析

Generation and Analysis of Transgenic Mice and Pigs Harboring the Cellulase and Phytase Genes Drived by Porcine Pancreatic Amylase Promoter

林育聖(Yu-Sheng Lin)

指導教授 ： 鄭登貴

國立臺灣大學/生物資源暨農學院/畜產學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.10289

摘要

本研究旨在將分別由本土水牛瘤胃真菌及牛糞便中分離出之纖維素分解酶（cellulase）、植酸分解酶（phytase）基因與由豬胰臟選殖出之具組織特異性的胰澱粉酶基因啟動子（pancreatic amylase promoter）構築而成的轉殖基因（pAMY-CEL、pAMY-PHY），藉由基因顯微注射及胚移置的技術，產製攜帶有該基因之轉基因小鼠與轉基因豬，冀望達到改進家畜對飼料中纖維素與有機磷的消化與利用，並增加生長效率、減少排泄物，進而達到環保與提升農民收益的最終目標。
在轉基因小鼠產製之試驗中，合計完成267個小鼠原核胚之基因顯微注射，其中245個經基因注射後外表完整且初步判定存活之小鼠胚，分別移置於10隻受胚母鼠，待懷孕期滿後陸續產下合計63隻仔小鼠。離乳後僅存活57隻，分別剪取尾巴組織抽取基因組DNA（genomic DNA）進行聚合酶連鎖反應（polymerase chain reaction）及南方吸漬法（Southern blot）分析，證實其中4隻小鼠確係帶有pAMY-CEL基因者；有6隻小鼠確係帶有pAMY-PHY基因。待此等轉基因小鼠發育達性成熟後，除與具生育力之ICR雄性或雌性小鼠進行配種，以瞭解轉殖基因小鼠的性腺傳承外，並同時收集其排泄物，進一步分析其對纖維素與有機磷的消化利用情形。
在轉基因豬產製之試驗中，合計完成145個豬原核胚之基因注入，其中138個業經完成基因注射後外表完整且存活之豬胚，並移置於6頭發情同期化之受胚豬後，合計獲得13頭仔豬出生；經分別抽取其基因組DNA進行PCR反應及Southern blot分析結果，證實其中5頭(3♀ /2♂)仔豬確係帶有pAMY-CEL基因者；2頭(1♀ /1♂)仔豬確係帶有pAMY-PHY基因。其中除編號Tg 13-2雌性仔豬不幸於出生後第10日即告夭折外，目前均已發育迄保育階段，刻正期待達性成熟，並分別與生育力正常之非轉基因豬完成配種，俾進一步分析確認轉殖基因在性腺之傳承效率，及其對纖維素與有機磷的消化利用情形。

關鍵字

環保豬 ； 植酸分解酶纖維素分解酶

並列摘要

Generation and Analysis of Transgenic Mice and Pigs Harboring the Cellulase and Phytase Genes Drived by Porcine Pancreatic Amylase Promoter
Yu-Sheng Lin
Abstract
The objective of this study was to generate transgenic animals carrying designed transgene(s) which includes a gastrointestinal track specific gene promoter and a structure genes derived from the fiber or phosphorous digestive enzymes of native water buffalo’s rumen microorganisms. Those transgenic animals are expected to have better ability to digest fibers, use the phosphorous more efficiently, produce less solid waste, and ultimately brought more profit to the farmers.
Base on the purpose described, we have cloned the promoter of porcine pancreatic amylase which would limit the secretion of target enzymes to the duodenum, avoiding the extreme pH value of stomach acid and retained the functions of proteins.
Using the technique of microinjection, the purified transgenes were delivered into the pronuclei of fertilized eggs that were surgically obtained from the donor animals. Those injected eggs then were transferred into the oviduct of the recipients.
To date, one hundred and forty five fertilized eggs were injected and transferred. There were two sows giving birth and thirteen piglets were born. Desired full-length transgenes were found in five of those piglets by the polymerase chain reaction (PCR) analysis. Further, the genomic DNA from these five piglets was extracted from ear pieces and was use in Southern blot analysis. Multiple bands were observed in most of the new born animals. It is possible that the transgenes were inserted as single copy and multiple copies which in the format of tail to tail or head to tail. Nonetheless, these results confirmed that piglets number 29-7 and 29-8 harboring transgenes, pAMY-PHY and pAMY-CEL; piglets number 29-3, 13-2, and 13-5 harboring single transgene pAMY-CEL.
For the experiments of producing transgenic mice, there are sixty three pups were born and genomic DNA was extracted from their tails and used in PCR analysis. There are four pups were found carrying the desired transgenes in full length. The Southern blot analysis is currently on going.

並列關鍵字

phytase ； cellulase

參考文獻

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