CN107043787A - 一种基于CRISPR/Cas9获得MARF1定点突变小鼠模型的构建方法和应用 - Google Patents
一种基于CRISPR/Cas9获得MARF1定点突变小鼠模型的构建方法和应用 Download PDFInfo
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Abstract
一种基于CRISPR/Cas9获得MARF1定点突变小鼠模型的构建方法和应用,设计高效的识别特定基因组PAM区域的sgRNA序列;并在sgRNA序列基础上设计Donor DNA;将Donor DNA,sgRNA和Cas9 mRNA混合物对受精***注射,胚胎移植,品系基因鉴定,得到founder鼠,并克隆测序,确认最终得到遗传性小鼠突变模型。本发明将可以对提高人工辅助生殖技术,防治女性***不育和女性生殖相关疾病,以及研制新型有效的女性避孕药物都具有重要参考价值,对促进我国乃至全球女性的生殖健康具有重大意义。
Description
技术领域
本发明涉及基因编码技术领域,具体涉及使用CRISPR/Cas9基因编码技术构建MARF1定点突变小鼠模型的方法和应用。
背景技术
MRAF1蛋白简介
苏等最近发现了一个编码过去功能未知的全新蛋白的Riken基因(4921513D23Rik)。携带该基因单位点突变和基因陷阱(gene trap)突变的小鼠均表现出同样的雌性不育而雄性正常的表性。进一步研究表明,导致雌性不育的直接原因是雌鼠***不能恢复减数***而排出未成熟的***。据此,我们将该基因命名为Marf1(meiosis arrest female 1)。在临床上,MARF1蛋白也具有重要的意义。在我们实验的基础上,陈子江等曾对卵子成熟障碍患者基因的关键外显子进行测序,探讨人类卵子成熟障碍是否与MARF1基因变异相关。
MARF1是一个RNA结合蛋白,除了拥有该类蛋白一般具有的RRM(RNA recognitionmotif)外,它在N-端拥有1个类似核糖核酸酶(RNase)的结构域(domain),在C-端拥有1个新被发现的、被命名为OST(oskar,tudor)或LOTUS(limkain,oskar and tudor domaincontaining proteins 5 and 7)的结构域。OST/LOTUS结构域也存在于果蝇蛋白Oskar和哺乳动物蛋白TDRD5和7(tudor domaincontaining proteins 5 and 7)中,并且被推测能够结合双链RNA特别是那些经与piRNA杂交后而形成的双链RNA,然后把它们携带到生殖细胞内诸如nuage或germ granule的特定部位。Oskar和TDRD5,7分别在果蝇生殖细胞决定(germcell specification)和小鼠雄性生殖细胞减数***及反转座子沉默中起重要作用(Su.et.al.PNAS.2012)。具体见图1。
MARF1蛋白定点突变
在蛋白质结构数据库NCBI-Structure(https://www.ncbi.nlm.nih.gov/ Structure/cdd/)进行保守的蛋白结构域类比,比较了NYN和同样保守的XNI,5’-EXONUCLEASE,TAQ DNA POLYMERASE,RIBONUCLEASE H,发现它们拥有4个保守的Asp核酸酶催化残基。在MARF1蛋白的NYN依次对应为D178,D215,D246,D272。在蛋白结构研究中,丙氨酸是氨基酸中侧链最短的手性氨基酸。通过将某氨基酸残基突变为丙氨酸,可以通过考察该突变蛋白的功能是否因此突变而失去或改变,从而可以定位对该蛋白功能有关键影响的氨基酸残基。置换成丙氨酸,去除了侧链上的活性基团,换成了体积小、无其他官能团的甲基,同时对蛋白质结构的影响较小。因此,本发明选择了突变272号位点,将天冬氨酸(D)突变为丙氨酸(A)。MARF1的272号天冬氨酸(D)突变为丙氨酸(A)后,可以用于具体研究MARF1的NYN结构域失活之后对整个MARF1蛋白的影响,进一步细致的研究MARF1蛋白丧失NYN酶活性后,对***转录后mRNA稳态调节的影响。具体序列比较见图2。
CRISPR/Cas9***简介
CRISPR(clustered,regularly interspaced,short palindromic repeats)是一种来自细菌降解入侵的病毒DNA或其他外源DNA的免疫机制。在细菌及古细菌中,CRISPR***共分成3类,其中Ⅰ类和Ⅲ类需要多种CRISPR相关蛋白(Cas蛋白)共同发挥作用,而Ⅱ类***只需要一种Cas蛋白即可,这为其能够广泛应用提供了便利条件。目前,来自Streptococcus pyogenes的CRISPR/Cas9***应用最为广泛。Cas9蛋白(含有两个核酸酶结构域,可以分别切割DNA两条单链。Cas9首先与crRNA及tracrRNA结合成复合物,然后通过PAM序列结合并侵入DNA,形成RNA-DNA复合结构,进而对目的DNA双链进行切割,使DNA双链断裂。由于PAM序列结构简单(5’-NGG-3’),几乎可以在所有的基因中都能找到大量靶点,因此得到广泛的应用。CRISPR/Cas9技术已经成功应用于植物、细菌、酵母、鱼类及哺乳动物细胞,是目前最高效的基因组编辑技术。通过基因工程手段对crRNA和tracrRNA进行改造,将其连接在一起得到sgRNA(single guide RNA)。融合后的RNA具有与野生型RNA类似的活力,但因为结构得到了简化,更方便研究者使用。具体见图3。
发明内容
解决的技术问题:本发明提供一种基于CRISPR/Cas9获得MARF1定点突变小鼠模型的构建方法和应用,在动物水平开展***优势表达的基因MARF1蛋白的研究。尤其是可以探究当发生点突变时,NYN结构域是否会失去活,以对MARF1如何调控***生长发育过程中的3个关键环节—“减数***恢复能力的获得”、“mRNA转录后调控”和“反转座子沉默”展开更加深入的研究。本发明预计可以首次揭示MARF1的NYN结构域作用原理,并将深入揭示调控***生长发育成熟的关键机制。这对提高人工辅助生殖技术,防治女性***不育和女性生殖相关疾病,以及研制新型有效的女性避孕药物都具有重要参考价值,对促进我国乃至全球女性的生殖健康具有重大意义。
技术方案:一种基于CRISPR/Cas9获得MARF1定点突变小鼠模型的构建方法,包括以下步骤:第1步:设计高效的识别特定基因组PAM区域的sgRNA序列,所述sgRNA序列如SEQID NO.1所示;并在sgRNA序列基础上设计Donor DNA,所述Donor DNA序列SEQ ID NO.2所示;第2步:将Donor DNA,sgRNA和Cas9 mRNA混合物对受精***注射,胚胎移植,品系基因鉴定,得到founder鼠,并克隆测序,确认最终得到的MARF1-D272A点突变的遗传性小鼠突变模型。
上述构建方法获得的基于CRISPR/Cas9获得MARF1定点突变小鼠模型在筛选避孕药物中的应用。
上述构建方法获得的基于CRISPR/Cas9获得MARF1定点突变小鼠模型在筛选防治女性***不育或女性生殖疾病药物中的应用。
获得MARF1定点突变小鼠模型的试剂盒,含有如SEQ ID NO.1所示的sgRNA。
上述试剂盒还含有SEQ ID NO.2所示的Donor DNA序列。
上述试剂盒还含有Cas9 mRNA。
转基因小鼠模型的具体构建过程如下:
1、CRISPR-Cas***元件的构建
在UCSC网站,下载MARF1的基因组DNA序列。进入http://crispr.mit.edu/网站,输入CDS序列查找可用的sgRNA,围绕MARF1-D272A点突变位置前后选择250bp。构建px330-sgRNA质粒,并制备sgRNA体外转录模板,体外转录得到sgRNA。进行Cas9 mRNA体外转录。
2、MARF1-sgRNA效率及Cas9 mRNA质量检测
使小鼠超数***后和公鼠合拢,检栓,获得更多的受精卵。配制sgRNA和Cas9 mRNA混合物。通过PCR,克隆,测序,来确定sgRNA切割位点的检测。
3、Donor DNA的设计,MARF1点突变小鼠的原核注射和胚胎移植
根据sgRNA切割位点来设计Donor DNA,合成ssDNA。配制donor DNA,sgRNA和Cas9mRNA混合物,原核注射,胚胎移植。
4、MARF1点突变小鼠的基因型鉴定
将新生小崽编号,并剪取部分脚趾提取基因组DNA。通过PCR,克隆,测序,来确定是否发生了MARF1-D272A点突变的精确重组。最终建立小鼠基因组中MARF1-D272A点突变的小鼠模型,出生的首代为founder小鼠;每个founder都将被视为一个独立的品系进行繁育,对每个founder品系都进行首次剪趾,二次剪尾的PCR鉴定,以及分别克隆并测序核实。MARF1蛋白NYN结构域的272位天冬氨基酸突变为丙氨酸,所述突变后的氨基酸序列如SEQ IDNO.1所示。
有益效果:(1)本发明提供的基于CRISPR/Cas9获得MARF1定点突变小鼠模型的构建方法,在实际应用中方便研究多功能MARF1在小鼠生殖发育尤其是***发育方面的作用机制,且该模型可以稳定传代。(2)人类卵子珍稀且受医学伦理限制不能轻易作为实验对象,“基因改造小鼠”是研究人类卵子的重要模型。本发明为临床女性***疾病的研究、产前诊断、第三代“试管婴儿”PGD(preimplantation genetic diagnosis),即移植前基因诊断、辅助生殖技术的进步以及避孕药物的开发提供了可稳定遗传的有效研究平台。
附图说明
图1.MARF1-NYN结构域保守位点序列比较图。
图2.MARF1-NYN结构域保守位点序列比较图。
图3.CRISPR-Cas9***简介图。
图4.MARF1-272-sgRNA-F1、MARF1-272-sgRNA-F2在基因组剪切效率图。
图5.MARF1-272定点突变Donor DNA序列,MARF1-272-sgRNA-F1,MARF1-272-sgRNA-F2在基因组的位置和对应的PAM区域,和相应的同义突变示意图。
图6.MARF1-D272A定点突变小鼠鉴定-PCR和StyI酶切图。
图7.MARF1-D272A定点突变小鼠鉴定-测序图。
图8.MARF1-D272A定点突变小鼠克隆后鉴定-PCR和StyI酶切图。
图9.MARF1-D272A定点突变小鼠克隆后鉴定-测序图。
图10.MARF1点突变雌性小鼠***与正常细胞对比图。
图11.MARF1点突变小鼠***与正常细胞检测指标对比图,图中每组数据的左侧数据柱为正常细胞,右侧数据柱为点突变细胞。
具体实施方式
本实验中需要的实验材料具体为:
Q5 High-fidelity 2XMaster Mix(NEB,M0492S)
2×Taq PCR Master Mix with loading dye(TIANGEN,KT201)
mMESSAGE mMACHINE T7 ULTRA kit(Ambion,AM1345)
MEGAshortscript TM T7ULTRA kit(Ambion,AM1354)
plasmid miniprep plus purification kit(genemark,Dp01-plus/Dp01-plus-300)
实施例1
一种基于CRISPR/Cas9获得MARF1定点突变小鼠模型的构建方法和应用,下面结合具体实施例,进一步阐述本发明。
1.1 CRISPR-Cas***元件的构建
1.1.1 用于基因敲入的sgRNA序列的设计
(1)进入UCSC网站,下载MARF1的基因组DNA序列,用SnapGene软件编辑序列。
(2)进入NCBI网站,根据基因信息在序列上标注外显子、内含子、启动子、编码序列(coding sequence,CDS)特征。
(3)进入http://crispr.mit.edu/网站,输入CDS序列查找可用的sgRNA,围绕MARF1-D272A点突变位置前后选择250bp。
(4)根据网站给出的分值选择sgRNA,分数越高代表其脱靶效应越低,sgRNA的特异性越好。同时也要综合考虑sgRNA在CDS上的位置,使用NCBI的domain architectureretrieval工具预测该基因CDS编码的蛋白有哪些重要结构域,尽量保证sgRNA位于结构域之前。
1.1.2 px330-sgRNA质粒构建
(1)px330质粒上带有U6启动子,其下游紧邻20碱基对(base pair,bp)的sgRNA,其后为约80bp的sgRNA通用骨架序列。将原始质粒用BbsⅠ单酶切后可用于构建新的质粒。
(2)合成寡聚脱氧核苷酸(DNA oligo,5’→3’)
sg-F:CACC-agtgatcttaggcaccggca
sg-R:AAAC-tgccggtgcctaagatcact
T7-sg-F:TAATACGACTCACTATAGGGagtgatcttaggcaccggca
T7-sg-R:AAAAGCACCGACTCGGTGCC
(3)oligo的退火(Anneal)
混匀,PCR仪中37℃30min;95℃ 5min;0.1℃/s降温至25℃
(4)退火后的片段连接到载体
混匀,室温反应3h,转化6μL至大肠杆菌感受态、涂板
(5)16h后挑菌,加200μL 2×YT培养基(Amp+)摇菌2h,菌液PCR
混匀,PCR反应:95℃热启动,58℃退火,延伸15s,35×Cycles。
(6)跑1%琼脂糖凝胶,PCR鉴定阳性的菌选3个送测序,测序引物为U6 promoter。
(7)选择测序结果正确的菌液小摇提质粒。
1.1.3 sgRNA体外转录模板的制备
(1)高保真酶PCR扩增编码sgRNA序列的DNA片段(约120bp),每条sgRNA配2管相同体系
混匀,PCR反应:98℃热启动,58℃退火,延伸12s,35×Cycles。
(2)配1%琼脂糖凝胶,跑胶,切下亮带大小120bp左右的胶块,使用GelExtraction试剂盒回收DNA。
a.胶块称重,100mg估算为100μL,加入3倍胶体积的Buffer QG。
b.65℃烘箱放置10min直至胶块融化。
c.加入1倍胶体积的异丙醇,颠倒混匀。
d.将混合物转移至吸附柱,13000rpm离心1min,弃滤液。
e.加入500μL Buffer QG,13000rpm离心1min,弃滤液。
f.加入750μL Buffer PE,13000rpm离心1min,弃滤液。
g.重复步骤f。
h.空管离心2min。
i.将吸附柱转移至干净1.5mL进口离心管,在RNA超净台中开盖吹干5min。
j.向吸附膜中央加入30μL 65℃预热的RNase-free ddH2O,室温静置5min。
k.13000rpm离心2min洗脱,吸出2μL测浓度(DNA50)。
1.1.4 sgRNA的体外转录
(1)(以下均在RNA超净台内完成)使用MEGAshortscriptTM T7转录试剂盒,配体外转录反应体系
混匀,37℃反应4h。
(2)加入0.5μL TURBO DNase,混匀,37℃反应15min。
(3)将体系转移至0.6mL小管,加入125μL RNase-free ddH2O和15μL AmmoniumAcetate Stop Solution,混匀。
(4)加入等体积下层酚氯仿,颠倒混匀10min,室温12000rpm离心10min。
(5)将上层水相转移至新管,加等体积氯仿,颠倒混匀10min,室温12000rpm离心10min。
(6)将上层水相转移至新管,加2倍体积无水乙醇,混匀,-20℃放置30min。
(7)配2%琼脂糖凝胶,用RNase-free ddH2O配75%乙醇,预冷4℃离心机。
(8)4℃ 12000rpm离心15min。
(9)弃上清,用500μL 75%乙醇漂洗一遍沉淀,4℃ 12000rpm离心5min。
(10)弃尽上清,RNA台中开盖吹干至沉淀的边缘趋于透明。
(11)视沉淀大小,加入50~80μL RNase-free ddH2O溶解,冰上放置2~3min。
(12)混匀,留3μL,剩余6μL/管分装,冻于-80℃冰箱。
(13)测浓度(ssDNA33),记录260/280(2.0~2.2为正常)。
(14)取1μL跑2%琼脂糖凝胶,130V 8min,检查条带亮度是否与浓度一致,条带大小是否在100bp左右。
1.1.5 Cas9 mRNA体外转录
(1)单酶切pSpCas9(BB)(pX330;Addgene plasmid ID:42230)质粒,作为体外转录模板
混匀,37℃反应过夜。
(2)配1%琼脂糖凝胶,跑胶,切下线性化DNA所在胶块,使用GelExtraction试剂盒回收。
(3)使用mMESSAGET7转录试剂盒体外转录Cas9 mRNA。
①(以下均在RNA超净台内完成)配体外转录反应体系
混匀,37℃反应2h。
②加入1μL TURBO DNase,混匀,37℃反应15min。
③配加polyA尾的反应体系
混匀,吸出2.5μL冰上保存。
④加入4μL E-PAP Mix,混匀,37℃反应30min,放于冰上。
⑤加入10μL Ammonium Acetate Stop Solution,混匀。
⑥酚氯仿抽提法进行RNA的纯化,步骤同于1.1.4(4)~(10)。
⑦视沉淀大小,加入Nuclease-free water溶解,冰上放置2~3min。
⑧混匀,取1.5μL测浓度(RNA40),260/280在2.0左右为正常,剩余mRNA暂放于冰上。
⑨根据测得浓度将mRNA稀释至约1μg/μL,取1μL以及加polyA尾前的2.5μL跑2%琼脂糖凝胶,正常情况下加polyA尾前应有明显亮带,加polyA尾后的条带应更大(可能略微模糊,不影响使用)。
⑩如果浓度、跑胶结果均正常,则1μg每管分装,迅速冻于-80℃冰箱,得Cas9mRNA。
1.2 MARF1-sgRNA效率及Cas9 mRNA质量检测
在一次实验中,可以用1条确定高效的sgRNA检测Cas9 mRNA的质量,也可用高质量Cas9 mRNA检测1条sgRNA的效率。
1.2.1 小鼠受精卵的获得
用于效率检测的胚胎一般取自BDF1品系小鼠,它是近交系C57BL/6J和DBA/2小鼠交配后培育的第一代杂交后代,6~8周性成熟。使用的小鼠均饲养在无特定病原体(specific-pathogen-free,SPF)。
(1)0d,13:00,抓取周龄8周左右的BDF1雌鼠,腹腔注射50U/mL(约60μL)的孕马血清***(pregnant mare’s serum gonadotropin,PMSG),模仿内源性促卵泡素(follicle-stimulating hormone,FSH)的促进卵成熟作用。
(2)2d,13:00,抓取注射过PMSG的BDF1雌鼠,腹腔注射50U/ml(约70μL)的人绒毛膜***(human chorionic gonadotropin,hCG),模仿促黄体素(luteinizinghormone,LH)的诱导***作用。并将每只雌鼠单独与1只BDF1公鼠合笼交配。
(3)做G1胚胎培养液滴:35mm Petri培养皿底,约20μL/滴,整齐排列,最后用Mineral oil封住液面,37℃,5%CO2培养箱中平衡过夜。
(4)3d,8:00,检查雌鼠有无***栓(公鼠交配后留下的分泌物痕迹),如有,则取出鼠房。
(5)做CZB胚胎培养液滴,37℃,5%CO2培养箱中至少平衡15min。
(6)颈椎脱臼法处死雌鼠:将小鼠放在鼠笼的铁架上,使其前爪抓住上面的钢丝条,紧紧压住它的颈部(头骨后),同时水平向后拉伸尾部,使其断颈。
(7)把处死的小鼠背部朝上放于搪瓷缸内,立即用75%酒精喷洒小鼠背部,尽量减少小鼠毛发的污染。
(8)捏住小鼠背部皮肤,用外科剪刀在中下区剪开一个小口,用力抓住剪开的皮肤,向头部和尾部拉伸使背部皮肤完全打开,毛发也完全离开内部组织。
(9)用眼科镊和锋利的剪刀打开背膜,用镊子夹起白色脂肪组织,轻轻地把子宫、输卵管、卵巢和脂肪垫整体拉出体腔,用镊子夹住卵巢下方的子宫,用剪刀在靠近输卵管的子宫系膜上刺一个洞,剪开子宫系膜,再用剪刀将卵巢与输卵管分离(确保输卵管的完整性),最后剪断靠近输卵管的子宫,将输卵管放入HCZB体外操作液。
(10)在解剖镜下用注射器的针尖刺破输卵管膨大的壶腹部,获得卵丘细胞包裹的受精卵团。
(11)收集所有团块,在0.1%透明质酸酶中37℃消化3~5min后,受精卵与卵丘细胞分离。
(12)在酒精灯的火焰上灼烧毛细玻璃管拉针并断针,内径大约150~200μm,组装口吸管。用口吸管移卵,分离出来的受精卵在HCZB滴中洗6遍,洗去残留的卵丘细胞和透明质酸酶。
(13)将受精卵移入CZB滴中洗3遍,37℃,5%CO2培养箱中恢复。
1.2.2 sgRNA和Cas9 mRNA混合物配制
混合物在RNA台配制。用于检测效率的混合物为100ng/μL的Cas9 mRNA和50ng/μL的sgRNA。一般注射需配制5μL混合物
Cas9 mRNA 500ng
sgRNA 250ng
ddH2O 补至5μL
4℃ 10000rpm离心10min,原核注射至约20枚受精卵中。
1.2.3 sgRNA切割位点的检测
A.胚胎的裂解
(1)受精卵在G1培养液中培养3天,发育至囊胚后用口吸管将胚胎移入5μL G1裂解液中。
(2)PCR仪中95℃ 20min裂解胚胎释放DNA。
(3)加入5μL G2溶液,混匀,可作为PCR模板。
B.PCR扩增
(1)用NCBI的Primer-BLAST工具设计特异性高的引物,PCR产物为小鼠基因组DNA上包含sgRNA位点的500bp左右片段,sgRNA位点应尽量在片段中央。
(2)用普通小鼠基因组DNA测试引物特异性,温度梯度PCR以得到引物最佳的退火温度。
(3)第一轮PCR反应
混匀,PCR反应:94℃热启动,延伸36s,30×Cycles。
(4)第二轮PCR反应,每个样品配2管相同体系;同时配2管以野生型(wild type,WT)小鼠基因组DNA为模板的体系
混匀,PCR反应:95℃热启动,延伸36s,35×Cycles。
(5)配1%琼脂糖凝胶,跑胶,切下条带正确的胶块,使用DNA纯化回收试剂盒回收DNA。
①胶块称重,100mg估算为100μL,加入等倍胶体积的Buffer PC。
②65℃烘箱放置约10min直至胶块融化。
③平衡吸附柱:加入500μL Buffer BL,13000rpm离心1min,弃滤液。
④将溶胶液加到吸附柱中,室温静置5min,13000rpm离心1min,弃滤液。
⑤加入600μL Buffer PW,13000rpm离心1min,弃滤液。
⑥重复步骤⑤。
⑦空管离心2min。
⑧将吸附柱转移至干净1.5mL离心管,在超净台中开盖吹干5min。
⑨向吸附膜中央加入30μL 65℃预热的Buffer EB,室温静置5min。
⑩13000rpm离心2min洗脱,吸出1.7μL测浓度(DNA50)。
C.胶回产物的T7酶切试验
(1)配退火反应体系
胶回收产物DNA 250~300ng
NEB Buffer 2 1.5μL
ddH2O 补至14.5μL
混匀,PCR仪中变性、退火反应:95℃ 5min,0.1℃/s缓慢降温至25℃,25℃ 10min。
(2)配2%琼脂糖凝胶。
(3)向DNA退火体系加入0.5μL T7内切酶Ⅰ,混匀,PCR仪中37℃反应15~17min。
(4)迅速加入10×Loading Buffer,混匀,135V电压跑胶30min,查看结果,如果除了约500bp主带外还有明显的偏小条带,而WT无,则说明原胶回收产物中包含了突变的片段,相应sgRNA可能有切割造成突变。
D.连接测序载体
(1)将T7酶切试验阳性的胶回片段连接测序载体,有两种商品化载体可用。
a.pLB vector
①配末端补平反应体系
混匀,PCR仪中20℃ 5min,70℃ 5min,短暂冰上。
②配连接反应体系
补平产物 4μL
pLB vector 0.5μL
T4Ligase 0.5μL
混匀,室温放置10min,全部至大肠杆菌感受态、涂板。
b.Zero Cloning
配连接反应体系
胶回收产物 25ng
T5 Zero Cloning Vector 1μL
ddH2O 补至4μL
混匀,室温放置15min,全部转化至大肠杆菌感受态、涂板。
(2)16h后挑菌,加200μL 2×YT培养基(Amp+)摇菌2h,菌液PCR
混匀,PCR反应:95℃热启动,延伸36s,35×Cycles。
(3)跑1%琼脂糖凝胶,PCR鉴定阳性的菌选8个送测序,测序引物为T7 promoter。查看结果,包含***/缺失(insertion/deletion,indel)突变的结果占总数的比例代表了相应sgRNA的效率。
(4)对于T7酶切试验阴性的样品,直接将胶回片段送测序,查看测序的峰图,如果在sgRNA切割位点之前都是单一的峰,而在sgRNA附近处开始至结尾有套峰,则该胶回产物可能包含了突变的片段(T7酶切试验假阴性),进行连接测序载体确认突变的存在。
1.3 Donor DNA的设计和MARF1点突变小鼠的原核注射
1.3.1 Donor DNA的设计
以sgRNA1的PAM区域5‘端方向3-4bp之间切割位置为中心,分别向5‘端方向65bp为左侧同源臂,3‘端方向65bp为右侧同源臂,订购单链ssDNA(生工,上海中国)。该单链ssDNA的纯度必须要采用HPLC纯化。
MARF1-272-sgRNA-1、MARF1-272-sgRNA-2的切割效率比较见图4。
MARF1-272donor DNA的设计图谱见图5。
1.3.2 donor DNA,sgRNA和Cas9 mRNA混合物配制
混合物在RNA台配制。用于检测效率的混合物为100ng/μL的Cas9 mRNA和50ng/μL的sgRNA,100ng/μL的donor DNA。一般注射需配制5μL混合物
4℃ 10000rpm离心10min,胞质注射至约受精卵中。
1.3.3 原核注射
受精卵的获得,和原核注射已描述,同上,见sgRNA效率及Cas9 mRNA质量检测。
1.3.4 胚胎移植
A、结扎公鼠的准备
(1)称重后按100ml/kg的剂量麻醉6-8周龄公鼠,背位固定小鼠,75%的乙醇涂搽消毒手术切口部位,以防止毛发污染切口。
(2)于生殖器前方大约1.3cm处横行剪开下腹部皮肤,作约1cm的切口,用浸75%乙醇的纱布搽拭切口部位以清理毛发。
(3)切开腹膜,进行膀胱定位。每侧都可见有一条管道走行,用镊子轻轻夹持左侧管道,提起部分使手术视野清晰可见,确定其为输精管。
(4)用镊子将输精管捏成U型,另一把镊子在酒精灯上加热,在两结扎点间烫断输精管,将结扎的输精管放入体内再进行另一侧结扎。
(5)两侧手术完毕后,用0-5号缝合线缝合内外术口。将小鼠放于37℃恒温台上直到苏醒。手术后小鼠饲养2周后确定手术是否成功。
(6)实验性饲养:将1-2只母鼠与输精管切除小鼠合笼饲养,次晨进行***栓检查。有***栓的母鼠可用栓做涂片,显微镜下观察是否有***。也可培养至次日取输卵管检查是否有二细胞胚胎。如果有***或有二细胞胚胎则公鼠结扎不成功。
B、受体鼠的准备
(1)同时挑选发情的ICR或CD-1与结扎公鼠合笼。
(2)次日清晨检查供体鼠和受体鼠是否见栓,见栓当天或2.5后用于胚胎移植。
C、输卵管移植
(1)准备好消毒的手术器械、缝合线等。
(2)称重后按200ml/kg的剂量对小鼠实施1.25%阿佛丁溶液麻醉,剪去小鼠背部被毛,75%酒精消毒后,在脊柱侧1cm,约与最后1肋骨平齐处,剪开皮肤,用镊子沿切口钝性分离皮肤肌肉,用镊子轻轻拉出卵巢、输卵管和子宫,夹住部分脂肪,置于鼠背后皮肤上。
(3)用移植针吸入20枚注射后存活的受精卵,吸入顺序为:M16培养液&空气&M16培养液&空气&受精卵,在保证胚胎完全吸入的情况下,尽量减少培养液的吸入量。
(4)将小鼠放在解剖镜下,用小号止血钳捏住脂肪垫,用眼科镊子将包裹卵巢的膜撕开一小口,暴露出输卵管伞部,***移卵针,将受精卵吹入输卵管内,输卵管壶腹部有明显的气泡表明移植成功。
(5)用钝镊子将脂肪垫、卵巢、子宫和输卵管一并送回腹腔,缝合腹膜及皮肤切口,移植好的受体鼠在温台上放置10min恢复后移送到屏障环境内饲养。
(6)移植后的小鼠做标记,注明移植时间、品系名称、胚胎数量。产仔后注明出生日期和出生数量,每天观察出生幼仔的存活情况。
1.4.MARF1点突变小鼠的基因型鉴定
RNA注射受精卵并移植到假孕母鼠输卵管中后,一般19~20天生育小鼠。小鼠出生6天以后,其趾间分开,可剪趾作编号标记,剪尾尖小块组织用于基因组DNA的抽提。
1.4.1 基因组DNA的酚氯仿抽提
(1)剪取组织放入离心管,加入300μL含有蛋白酶K的鼠尾裂解液,65℃裂解过夜。
(2)加入300μL酚氯仿,颠倒混匀10min,室温12000rpm离心10min。
(3)小心吸取上清并移至新管,加入两倍体积的无水乙醇,颠倒混匀可见絮状沉淀,4℃12000rpm离心5min。
(4)弃上清,加入500μL 70%乙醇漂洗沉淀,4℃ 12000rpm离心5min。
(5)弃尽上清,超净台吹干5min至底部沉淀变透明,视沉淀大小,加入65℃预热的ddH2O。
(6)室温放置5min使DNA充分溶解。
(7)颠倒混匀,吸出2μL测浓度(DNA50)。
1.4.2 包含预测突变位点片段的PCR扩增
(1)使用的引物与sgRNA效率检测时的相同,每个DNA样本视引物效率配制2~3管反应体系
(2)胶回收,步骤同3.2.3B.(5)。
1.4.3 基因组PCR产物直接StyI酶切试验及测序确认
(1)PCR产物直接StyI酶切试验:
(2)37度,水浴,酶切3h,电泳。
(3)PCR产物直接测序。
由于定点突变的改变,如果发生精确的重组,将会增加一个酶切位点StyI。野生型的小鼠则不能够被切开。本发明出生的小鼠,#1可以被StyI酶切,但#2,#3,#4,#5都不可以被酶切。详细见图6。为了进一步确认发生精确的重组,测序可以发现重组的点突变位置会出现一个显著的双峰。而野生型的小鼠则不会出现双峰。本发明出生的小鼠,#1有双峰,但#2,#3,#4,#5都没有双峰。详细见图7。
1.4.4 PCR产物克隆后StyI酶切试验及测序确认
PCR产物通过QIANGEN试剂盒胶回收之后,用PLB vector试剂盒连接,克隆,挑菌,单克隆,做菌液PCR,筛选出阳性菌。一方面,将PCR产物直接用StyI酶切,电泳,单克隆菌株L5、L9可以被StyI酶切,但单克隆菌株L1、L2、L3、L4、L6、L7、L8都不可以被StyI酶切,见图8。另一方面,将单克隆阳性菌送去公司测序,测序结果见图9。
1.5 MARF1点突变小鼠作为稳定遗传模型研究雌性***的应用
1.5.1 MARF1点突变雌性小鼠***,***不能正常发育
如图10所示。MARF1点突变小鼠***阻滞在生发泡(GV)时期,不能够恢复减数***,不能正常成熟。进一步导致雌性***。
1.5.2 MARF1点突变小鼠***系列检测指标RNA显著上升
如图11所示。MARF1点突变小鼠***中大量的检测指标RNA显著上升,而正常***中大量的检测指标RNA维持较低水平。这些可以为产前诊断、第三代“试管婴儿”PGD(preimplantation genetic diagnosis),即移植前基因诊断、辅助生殖技术的进步以及避孕药物的开发提供了可稳定遗传的有效研究平台。
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Claims (6)
1.一种基于CRISPR/Cas9获得MARF1定点突变小鼠模型的构建方法,其特征包括以下步骤:
第1步:设计高效的识别特定基因组PAM区域的sgRNA序列,所述sgRNA序列如SEQ IDNO.1所示;并在sgRNA序列基础上设计Donor DNA,所述Donor DNA序列SEQ ID NO.2所示;
第2步:将Donor DNA,sgRNA和Cas9 mRNA混合物对受精***注射,胚胎移植,品系基因鉴定,得到founder鼠,并克隆测序,确认最终得到的MARF1-D272A点突变的遗传性小鼠突变模型。
2.权利要求1 所述构建方法获得的基于CRISPR/Cas9获得MARF1定点突变小鼠模型在筛选避孕药物中的应用。
3.权利要求1 所述构建方法获得的基于CRISPR/Cas9获得MARF1定点突变小鼠模型在筛选防治女性***不育或女性生殖疾病药物中的应用。
4.获得MARF1定点突变小鼠模型的试剂盒,其特征在于含有如SEQ ID NO.1所示的sgRNA。
5.根据权利要求4所述的试剂盒,其特征在于还含有SEQ ID NO.2所示的Donor DNA序列。
6.根据权利要求5所述的试剂盒,其特征在于还含有Cas9 mRNA。
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