表現紅色螢光蛋白質迷你豬之育成及其粒線體DNA分析

Abstract

自從發現來自珊瑚的Discosoma striata開始，這個通稱DsRed的螢光蛋白質便被大量應用至生物醫學等相關試驗研究上。DsRed的光譜範圍在500 ~ 583 nm之間，而其波峰在558 nm。紅色螢光蛋白因具有快速成熟、在動物體內辨識度較高以及對於pH值具有較佳的容忍力等優點，因此適合應用於配合其他螢光蛋白之多重顏色標定等實驗上。在先前的研究中，我們透過原核胚顯微注射已成功產製出帶有單體紅色螢光蛋白質基因之轉殖家豬。雖然家豬(Sus scrofa domesticus or Sus domesticus)可做為一種優秀的實驗動物，然其平均體型實驗操作而言仍過於龐大，6月齡時即可達到90公斤，相較於其他較小型實驗動物例如嚙齒類之維持成本以及體型龐大所帶來之試驗操作不便等問題，皆為以大型家豬為實驗動物尚有許多改進空間。因此，近年來以較小型豬隻如蘭嶼豬作為人類疾病相關研究之模式動物，有逐年上升之趨勢。此外，在大部分多細胞有機體中，粒線體DNA皆為母系遺傳，粒線體DNA被廣泛地運用於種源鑑定以及物種間基因親緣關係之判定。本研究之目的為表現紅色螢光蛋白質迷你豬之育成及其粒線體DNA分析。 本研究中，帶有單體紅色螢光蛋白質基因之洋種豬公豬與台灣大學育成之李宋品系母豬被用來雜交，從而產生表現紅色螢光蛋白質之小型化子代螢光豬。採集子代之細胞樣本並分離出DNA，經由PCR分析三組引子對序列之產物: DsRed (780bp)為單體紅色螢光蛋白質基因之偵測、mtDNA控制區為粒線體DNA中最具多型性之區域以及細胞色素-b (1308bp)為種間以及種內之種源關係鑑定之常用序列。 經PCR後之DsRed分析結果，顯示G1, G2, G3及G4之子代皆部分帶有單體紅色螢光蛋白質基因，且平均之性腺傳承率為67%。兩組具有蘭嶼豬專一性之粒線體DNA引子對分析結果則顯示包括G1, G2, G3及G4等各子代分別皆為蘭嶼品系之延伸。以上結果顯示子代不管是否帶有單體紅色螢光蛋白質基因，其皆證實為蘭嶼品系之延伸；G1到G3之子代在20週齡之平均體重有穩定下降之趨勢，其中G2之體重較G1下降76%，而G3則較G2之平均體重下降59%，以上結果意味著我們已經成功將表現紅色螢光蛋白質之轉殖豬迷你化，而進一步比對G3以及蘭嶼豬之間的體型參數 (8週齡與20週齡之體長、體高、體重) 則顯示G3已經符合”迷你豬”的標準範圍。 綜觀上述，本研究論文除成功產出表現紅色螢光蛋白之蘭嶼品系基因轉殖豬外，此等表現紅色螢光蛋白之迷你豬具有可提供作為生物醫學研究之模式動物之潛能。

DsRed, an Anthozoa-derived fluorescent protein with an excitation spectrum at 500 ~ 583 nm and a major peak at 558 nm has become a widely applied tool in the field of biomedical research. It has advantages such as faster maturation, less autofluorescence in animal tissues and better resistance to pH, making it ideal for multi-label colouring and dual-colour experiments with other fluorescent proteins. With the application of pronucleus injection, we were able to generate pigs harbouring the Ds-Red monomer protein. However, although the domestic pig (Sus scrofa domesticus or Sus domesticus) represents a great model animal, the size and weight of a regular pig (90 kg at 6 months), the expenses compared to smaller animals such as rodents and the difficulty of manipulation still pose a disadvantage. Hence, using smaller pigs such as the Lanyu-pig as an animal for human disease model has increased in numbers during the recent years. In most multicellular organisms, mtDNA is inherited from the mother (maternally inherited). Mechanisms for this include simple dilution, degradation of sperm mtDNA in the fertilized egg, and, at least in a few organisms, failure of sperm mtDNA to enter the egg. Whatever the mechanism is, this single parent (uniparental) pattern of mtDNA inheritance is found in most animals. In recent years, mtDNA has been widely used for phylogenetic studies to estimate the genetic relationship among mammalian species. The objective of this study is to breed a new strain of miniature pigs harbouring the DsRed monomer protein and the analysis of the mitochondrial DNA. For this study, male European domesticated pigs expressing the DsRed protein and female Lanyu-derived strain were utilized to breed a cross-hybrid offspring of miniature pigs expressing the DsRed monomer protein. Fibroblast cells of each generation was cultured for further total DNA extraction and three sets of primers were designed for PCR analyses: DsRed (780 bp), for detection of the DsRed monomer, mtDNA Control Region (1380 bp), which is an area of non-coding DNA and is the most polymorphic region of the mtDNA genome, and mtDNA cytochrome B (1308 bp), which is commonly used to determine phylogenetic relationships within families and genera. PCR analysis using the DsRed primer show that some of the offspring from the four relating generations G1, G2, G3 and G4 harbouring the DsRed monomer protein gene, with an average germ-line transmission rate at 67%. The two pairs of mtDNA primers, which are Lanyu specific, showed that the progenies to be Lanyu derived, whether or not harbouring the DsRed monomer protein gene. Steady decrease of body weight at 20 weeks age from G1 to G3, with G2 showing a 76% decrease in body weight compared to G1, and G3 showing a 59% decrease in body weight compared to G2, imply the Ds-Red transgenic line was successfully miniaturized. Further compare the physiological parameter (body length, height, weight, at 8th and 20th week) between G3 and Lanyu pig indicates that G3 offspring fit the description of a “mini-pig”. Taken above, the data show successful breeding of Lanyu-derived pig expressing the DsRed monomer protein, indicating this new breed of DsRed-miniature pigs possess potentials for use as a new model animal in the field of biomedical studies and application.