MSTN基因突变纯合子兔的繁育和表型分析

Propagation and phenotypic analysis of mutant rabbits with MSTN homozygous mutation

摘要:

肌肉生长抑制素（myostatin，MSTN）基因是动物骨骼肌生长的负调控基因。本研究在获得MSTN基因双等位突变原代兔的基础上，培育具有“双肌”表型性状和稳定遗传的MSTN^–/–纯合突变兔，建立原代MSTN双等位突变兔繁育纯合子后代的方法。应用CRISPR/Cas9基因编辑技术制备MSTN^–/–突变原代兔，原代双等位突变兔与野生型兔配种，产生F1代兔。同一突变系的F1代兔半同胞交配或回交繁殖F2代纯合子兔；后代兔利用基因组DNA PCR扩增产物序列分析和T载体克隆测序鉴定用于筛选纯合子兔；14–19周龄MSTN基因突变兔称量体重。MSTN基因突变兔臀大肌组织切片，统计分析肌纤维横截面积。获得5只MSTN基因突变原代兔，其中3只用于纯合子培育，共获得15只（5种突变子）纯合突变兔（M2-a：3只；M2-b：2只；M3-a：2只；M7-a：6只；M7-b：2只）。在14–19周龄，MSTN^–/–纯合突变兔体重极显著高于同龄MSTN^+/+野生型兔（平均（2 718±120） g和（1 969±53） g，P<0.01，同龄体重提高38.0%）；纯合突变兔的臀大肌纤维平均截面积极显著高于野生型兔（（3 512.2±439.2）μm²和（1 274.8±327.3）μm²，P<0.01），也显著高于MSTN^+/–半合子兔（（3 512.2±439.2）μm²和（2 610.4±604.4）μm²，P<0.05）。本研究成功培育了5种MSTN^–/–基因纯合突变兔，且有明显瘦肉表型。研究表明，应用CRISPR/Cas9基因编辑获得的原代双等位突变兔为非嵌合体突变兔，突变性状遗传后代；原代双等位突变兔后代分离为两个不同突变子，并都呈现“双肌”表型；F1代单等位突变兔通过近交或回交可获得MSTN^–/–纯合突变兔。该研究在利用基因编辑技术培育优良家畜品种方面取得了实验进展。

Abstract:

Myostatin gene (MSTN) encodes a negative regulator for controlling skeletal muscle growth in animals. In this study, MSTN^-/- homozygous mutants with "double muscle" phenotypic traits and stable inheritance were bred on the basis of MSTN gene editing rabbits, with the aim to establish a method for breeding homozygous progeny from primary MSTN biallelic mutant rabbits. MSTN^-/- primary mutant rabbits were generated by CRISPR/Cas9 gene editing technology. The primary mutant rabbits were mated with wild type rabbits to produce F1 rabbits, whereas the F2 generation homozygous rabbits were bred by half-sibling mating or backcrossing with F1 generation rabbits of the same mutant strain. Sequence analysis of PCR products and its T vector cloning were used to screen homozygous rabbits. The MSTN mutant rabbits with 14-19 week-old were weighed and the difference of gluteus maximus tissue sections and muscle fiber cross-sectional area were calculated and analyzed. Five primary rabbits with MSTN gene mutation were obtained, among which three were used for homozygous breeding. A total of 15 homozygous rabbits (5 types of mutants) were obtained (M2-a:3; M2-b:2; M3-a:2; M7-a:6; M7-b:2). The body weight of MSTN^-/- homozygous mutant rabbits aged 14-19 weeks were significantly higher than that of MSTN^+/+ wild-type rabbits of the same age ((2 718±120) g vs. (1 969±53) g, P<0.01, a 38.0% increase). The mean cross sections of gluteus maximus muscle fiber in homozygous mutant rabbits were not only significantly higher than that of wild type rabbits ((3 512.2±439.2) μm² vs. (1 274.8±327.3) μm², P<0.01), but also significantly higher than that of MSTN^+/- hemizygous rabbits ((3 512.2±439.2) μm² vs. (2 610.4±604.4) μm², P<0.05). In summary, five homozygous mutants rabbits of MSTN^-/- gene were successfully bred, which showed a clear lean phenotype. The results showed that the primary breeds were non-chimeric mutant rabbits, and the mutant traits could be inherited from the offspring. MSTN^-/- homozygous mutant rabbits of F2 generation could be obtained from F1 hemizygous rabbits by inbreeding or backcrossing. The progenies of the primary biallelic mutant rabbits were separated into two single-allelic mutants, both of which showed a "double-muscle" phenotype. Thus, this study has made progress in breeding high-quality livestock breeds with gene editing technology.