以CRISPR-Cas9 系統產製肌肉生長抑制素基因編輯小鼠

Abstract

肌肉生長抑制素（myostatin）為乙型轉化生長因子（transforming growth factor beta）蛋白質家族的一員，myostatin 在體內主要功能為骨骼肌的負調控蛋白，當myostatin存在時會抑制肌肉生長。在1997年McPherron等人產製出myostatin null小鼠且發現其骨骼肌含量有明顯增加，此後便有許多對於myostatin應用之研究，例如:抑制myostatin挽救因疾病而造成之肌肉萎縮，或是將動物體之myostatin突變使產肉率增加，但上述之應用方法都有其副作用，因此，本研究擬尋找較溫和之方法抑制myostatin，期能增加myostatin之應用性。本試驗預計將小鼠myostatin之基因進行點突變以產出兩種品系之myostatin基因編輯小鼠，分別為將小鼠myostatin第100個胺基酸由aspartate(D)突變為alanine(A)及第264~267之胺基酸由arginine-serine-arginine-arginine (RSRR)突變為glycine-leucine-aspartate-glycine (GLDG)，預期此兩種突變將分別造成myostatin前驅蛋白無法被bmp-1及furin convertase 蛋白酶進行水解使得myostatin 之配體無法產生，而導致myostatin無法活化下游訊號進而造成小鼠肌肉量增加。CRISPR/Cas9基因系統能對目標基因產生雙股斷裂，提供外源基因當作修補斷裂之模板股便可以達到精準之基因編輯，且CRISPR/Cas9設計上較為方便因此被廣泛使用，本試驗首先對候選之sgRNA進行效率分析，選擇最適當之sgRNA進行myostatin基因編輯小鼠產製，利用小鼠胚原核注射的方式將sgRNA、ssodn模板股以及Cas9蛋白注入小鼠之原核，並將存活之胚移置進入假孕母小鼠輸卵管，待仔小鼠生下後進行基因型及表現型鑑定。本試驗成功產製出BMP-1水解點位突變之myostatin基因編輯小鼠，但目前仍只有基因型上之鑑定成功且為異型合子，未來將進行配種、增加小鼠族群數量並且配種出同型合子之小鼠，比較此種myostatin基因編輯小鼠之身體組成及myostatin與野生型小鼠有無差異，以期未來能對疾病所造成肌肉萎縮病人之治療研究提供參考方向，亦可能應用在經濟動物的生產上。

Myostatin is a member of the transforming growth factor beta protein family. The main function of myostatin in the body is a negative regulatory protein of skeletal muscle. When myostatin expressed, it can inhibit skeletal muscle growth. In 1997, McPherron et al. produced myostatin null mice and found that their skeletal muscle content increased significantly compared to skeletal muscle content of wild type mouse. Since then, there have been many studies on the application of myostatin, such as inhibiting myostatin to rescue muscle atrophy caused by disease, or mutate the myostatin in animals to increases the meat production. But the application mentioned above have their side effects. Therefore, this study intends to find a milder method to inhibit myostatin, hoping to increase the applicability of myostatin.This experiment is expected to point-mutated myostatin gene of mouse to produce two strains of myostatin gene-edited mice. One is mutated the 100th amino acid of mouse myostatin from aspartate (D) to alanine (A). The other is mutated the 264~267th amino acid from arginine-serine-arginine-arginine (RSRR) to glycine-leucine-aspartate-glycine (GLDG). Expect that these two mutations would cause the myostatin precursor protein to fail to be hydrolysis by bmp-1 protein and furin convertase, respectively. This would cause less myostatin ligand produced, then the myostatin signal pathway would not be activated causing skeletal muscle content increase in mouse. The CRISPR/Cas9 gene editing system can produce double-strand breaks in the target gene. By providing exogenous genes as template strands to repair the breaks, precise gene editing can be achieved. CRISPR/Cas9 is more convenient in design than previous nuclease and therefore widely used. The candidate sgRNAs would be chosen to test their gene editing efficiency at first. Then chose the appropriate sgRNA to produce myostatin gene edited mice. The sgRNA, ssodn template s and Cas9 protein were injected into the pronucleus of the mouse by pronuclear injection, and the surviving embryos were transferred into the oviducts of pseudopregnant mouse. Genotype and phenotype identification were performed after the newborns were birth. In this experiment, the myostatin BMP-1 hydrolysis site mutation mouse had been produced, but currently only the genotype had been successfully identified and were heterozygous. In the future, need to increase the population of transgenic mouse and breed homozygous mouse. To compare the biological composition of this myostatin gene edited mouse and whether myostatin is different from wild-type mice. Hope it can provide a reference direction for the treatment and research of patients with muscle atrophy caused by diseases, and might also be applied to economic animal in the future.