本文關(guān)鍵詞：鼠fscb基因靶向敲除載體的構(gòu)建及動(dòng)物模型的建立　出處：《第三軍醫(yī)大學(xué)學(xué)報(bào)》2015年15期 　論文類型：期刊論文

  更多相關(guān)文章： 基因敲除 精子蛋白 CRISPR/Cas 動(dòng)物模型

【摘要】：目的利用CRISPR/Cas9系統(tǒng)構(gòu)建鼠fscb基因打靶載體,為研究鼠FSCB蛋白在精子鞭毛運(yùn)動(dòng)和獲能中作用建立fscb基因敲除動(dòng)物模型。方法根據(jù)fscb基因全長(zhǎng)序列,設(shè)計(jì)CRISPR/Cas9作用靶點(diǎn),合成3對(duì)單鏈向?qū)NA(single-guide RNA,sgRNA),并克隆進(jìn)PU57-T7-GDNA載體,通過(guò)PCR測(cè)序鑒定,獲得sgRNA表達(dá)質(zhì)粒。利用T7 RNA聚合酶體外轉(zhuǎn)錄sgRNA和Cas9 RNA。將Cas9mRNA和sgRNA以適當(dāng)比例混合后注射入B6J小鼠受精卵中,獲得fscb基因敲除的初建鼠(F0)。通過(guò)PCR對(duì)基因突變情況進(jìn)行鑒定,選取靶基因敲除的小鼠進(jìn)行傳代并進(jìn)一步測(cè)序分析確定突變情況。結(jié)果成功構(gòu)建表達(dá)sgRNA的載體并進(jìn)行體外轉(zhuǎn)錄,將有活性的sgRNA和Cas9 RNA直接注射入鼠受精卵,獲得39只F0代陽(yáng)性小鼠,選取3只明確刪除目的基因片段并產(chǎn)生移碼的雄鼠與野生雌鼠回交繁育得到5只F1代陽(yáng)性小鼠(2只雄鼠,3只雌鼠),將F1代雜合子雄鼠與雌鼠交配,獲得純合子小鼠。通過(guò)鑒定,建立了兩種不同基因型的fscb基因敲除鼠系,并傳代繁育。Western blot分析證實(shí)兩種鼠系純合子雄鼠睪丸及精子內(nèi)的FSCB蛋白表達(dá)消失。結(jié)論通過(guò)CRISPR/Cas9系統(tǒng)靶向敲除fscb基因并獲得純合子小鼠,為進(jìn)一步分析FSCB蛋白的功能提供了基因敲除小鼠模型。
[Abstract]:Objective to construct a mouse FSCB gene targeting vector by CRISPR/Cas9 system, for the study of mouse FSCB protein in the role of the establishment of the FSCB gene knock-out animal model of sperm flagellar motility and capacitation. Methods according to the sequence of the full-length FSCB gene design targets of CRISPR/Cas9, the synthesis of 3 of single wizard RNA (single-guide RNA, sgRNA), and cloned into PU57-T7-GDNA vector by PCR, sequencing, sgRNA expression plasmid. The use of T7 RNA polymerase in vitro transcription of sgRNA and Cas9 RNA. Cas9mRNA and sgRNA mixed in a proper ratio after B6J injected into mouse fertilized eggs, FSCB gene knockout mice (F0). First built by PCR to identify the gene mutation, selection of target gene knockout mice were sequenced and further analysis to determine the mutation. The expression vector sgRNA were constructed successfully and in vitro transcription, will have a direct injection of sgRNA and Cas9 in rat RNA activity The fertilized eggs, obtained 39 F0 positive mice, a total of 3 clear and delete gene frameshift in male rats and female rats received 5 backcross breeding wild F1 generation positive mice (2 males, 3 females), F1 heterozygous male and female mice mated to obtain homozygous mice. Through the identification, established FSCB gene two genotypes of knockout mice, and cultured breeding of.Western blot analysis confirmed that two mice homozygous male mice testis and sperm in the expression of FSCB protein disappeared. Conclusion through CRISPR/Cas9 system targeting FSCB gene knockout and homozygous mice, providing knockout in addition to a mouse model for further analysis of the function of FSCB protein.

【作者單位】： 第三軍醫(yī)大學(xué)大坪醫(yī)院野戰(zhàn)外科研究所泌尿外科;
【基金】：國(guó)家自然科學(xué)基金面上項(xiàng)目(81170617)~~
【分類號(hào)】：R-332
【正文快照】： 已有研究發(fā)現(xiàn)嚴(yán)重弱精癥或精子完全無(wú)運(yùn)動(dòng)的患者存在精子纖維鞘發(fā)育不良[1],精子形成過(guò)程中睪丸特異性基因的適時(shí)有序表達(dá),并驅(qū)動(dòng)精子各細(xì)胞器的有序形成包括精子鞭毛骨架結(jié)構(gòu)和相關(guān)蛋白的有序裝配整合是保證精子發(fā)揮正常功能的根本物質(zhì)基礎(chǔ)[2]。纖維鞘CABYR結(jié)合蛋白(fibrous

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 顏雯;李海濤;向華;王曉虎;;CRISPR-Cas基因組改造技術(shù)研究進(jìn)展[J];廣東農(nóng)業(yè)科學(xué);2014年02期

2 韋玲靜;黃sッ，

本文編號(hào)：1391300