以CRISPR-Cas9系統剔除小鼠UCP1基因對於生殖表現的影響

Abstract

小鼠為最常使用的模式生物之一，幼鼠的死亡會造成實驗室資源的損耗以及實驗進度的干擾。小鼠在斷奶之前的死亡率與母鼠哺乳期間的產乳量有顯著相關性，並根據散熱極限 (heat dissipation limit) 理論，體熱嚴重地影響了小鼠哺乳期的產乳表現，較高的環境溫度及體溫皆會使小鼠的產乳量下降，而溫度的降低及散熱能力的增加能增進小鼠產乳的能力，因此推測減少小鼠的產熱可能有助於提升小鼠哺乳期的產乳表現，以增加幼鼠的成長及存活率。 棕色脂肪組織為產熱功能最活躍的組織，其產熱透過解偶聯蛋白質1 (uncoupling protein 1, Ucp1) 來調節，亦有研究觀察到在哺乳期間UCP1的基因表現量有下降的情形。因此本試驗擬使用CRISPR-Cas9基因編輯系統，將小鼠UCP1基因之外顯子2及部分外顯子3之間序列做剔除，以使棕色脂肪組織之產熱功能失活，期望能使小鼠之產乳表現上升。本試驗首先以細胞試驗測試候選sgRNA之基因編輯可行性，再使用顯微注射技術將選用之sgRNA與Cas9蛋白質注射入小鼠一細胞期胚胎之原核內，以達到基因編輯的效果，透過胚胎移置方式使代孕母鼠產下目標基因編輯小鼠，以定序方式分析其序列與基因型，並使用西方墨點法對小鼠棕色脂肪組織做 Ucp1 蛋白質定量分析，隨後比較UCP1基因剔除小鼠之產乳量與野生型小鼠在哺乳期間的產乳量差異。 本試驗成功產製出 UCP1 雙等位基因剔除(UCP1-/-)小鼠，並且在其棕色脂肪組織上無測得Ucp1蛋白質的含量，顯示此基因剔除小鼠之棕色脂肪組織已喪失產熱功能，但在產乳量的測量上尚未觀察到與野生型小鼠有顯著的差異，推測可能與試驗小鼠之品系及攝食量差異相關。因此後續可對不同品系之小鼠進行相同之試驗測試，或增加限制攝食量之試驗變因，期望能觀察到產乳表現優於野生型小鼠之結果，使其有利於小鼠的繁殖及育種，並可將其基因功能特性供其他物種做為參考。

Mice are one of the most commonly used model organism, and the mortality of pups can lead to resource loss and disruptions in experimental progress in the laboratory. The mortality rate before weaning in mice is significantly correlated with the lactation performance of the mother mouse. According to the heat dissipation limit theory, body heat significantly influences lactation performance during the mouse lactation. Higher environmental temperatures and body temperatures both can reduces milk production in mice, while lowering the temperature and increasing heat dissipation capacity enhance lactation ability. Therefore, reducing the thermogenic capacity of mice may contribute to enhancing lactation performance during the mouse lactation, thereby increasing the growth and survival rates of the offspring. Brown adipose tissue is the most active tissue in thermogenesis, and its heat production is regulated by uncoupling protein 1 (Ucp1). Some studies have also observed a decrease in UCP1 gene expression during lactation. Therefore, this study plans to use the CRISPR-Cas9 gene editing system to delete the sequence between exon 2 and part of exon 3 of the mouse UCP1 gene, and it will result in the inactivation of the thermogenic function of brown adipose tissue and expect an increase in lactation performance in mice. The experiment starts with a cell test to select the available sgRNA sequences. Subsequently, the selected sgRNA and Cas9 protein are injected into the pronucleus of one-cell stage mouse embryos using microinjection technology to achieve gene editing. Target gene-edited mice are obtained through embryo transfer by surrogate mothers, and their sequences and genotypes are analyzed through sequencing. Then use the Western blotting to quantify Ucp1 protein in mouse brown adipose tissue and then compared the difference in milk production between UCP1 knockout mice and wild-type mice during lactation. In this experiment, UCP1 knockout (UCP1-/-) mice had been generated, and no Ucp1 protein was detected in the brown adipose tissue, indicating that the brown adipose tissue of UCP1 knockout mice has lost its thermogenic function. but no significant differences in milk production were observed compared to wild-type mice. It is speculated that it is related to differences for trials in mouse strains and food intake. Therefore, the same experimental test can be carried out on different strains of mice in the future, or the experimental variables of limiting food intake can be added. It is expected that the milk production performance of wild-type mice may be observed to be better than that of wild-type mice, which will be beneficial to the reproduction and breeding of mice. Additionally, the genetic characteristics of these mice could serve as a reference for other species.