CN117025687A - 一种转基因不育鸡的制备方法 - Google Patents
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Abstract
本发明公开了一种转基因不育鸡的制备方法,属于基因工程领域。上述方法包括以下步骤:(1)使用CRISPR/cas9介导的同源重组修复将HSV‑TK基因定点插入到鸡原始生殖细胞特异性表达DAZL基因最后一个编码外显子的3’端。使得所述原始生殖细胞特异表达所述HSV‑TK基因,回注受体鸡胚中,制备HSV‑TK嵌合体鸡;(2)经HSV‑TK嵌合体鸡配种之后得到HSV‑TK受体鸡胚,给予更昔洛韦能够消除内源生殖细胞,获得转基因不育鸡胚。可见,本发明通过HSV‑TK基因插入鸡原始生殖细胞,结合更昔洛韦特异性消除鸡胚中的内源原始生殖细胞,进而可作为原始生殖细胞移植的受体鸡胚,即获得转基因不育鸡模型。
Description
技术领域
本发明涉及基因工程领域,特别是涉及一种转基因不育鸡的制备方法。
背景技术
提高原始生殖细胞(Primordial germcells,PGCs)的细胞移植生殖系遗传效率的策略主要分为两种,其中一个策略是改进和优化PGCs的细胞培养体系,使得细胞在体外培养和基因操作之后仍然能否保持完整的生殖细胞特性,提高生成配子的能力,该方面的尝试包括缩短体外培养时间、添加促进生长和维持生殖细胞特性的物质等,但是效果不稳定,或者操作难度大,此外,该方式使得宿主胚胎的性腺同时包含引入的供体PGCS和内源性宿主PGCS,减少了随后交配的后代来自供体PGCS的机会,即生殖系遗传效率低。另外的一种策略是消除受体鸡胚内源的生殖细胞,提高性腺中移植的外源生殖细胞的比例,该方法包括利用物理辐射或者利用基因修饰定向消除内源生殖细胞。虽然通过以上方式,这些遗传上不育的受体宿主被证明能够有效地从移植的外源生殖细胞产生后代,但是均存在一些缺陷和不足。比如:单独的物理辐射方式,对发育中的受体宿主胚胎和成熟的宿主动物可能存在高度毒性;而通过消除生殖细胞发育所需基因进而消融内源性生殖细胞的手段,仅能制备雌性的不育鸡胚,不能用雄性PGCs移植。因此,急需进一步的优化和研究基于生殖细胞提高转基因生殖细胞嵌合体的效率的方法。
发明内容
本发明的目的是制备一种转基因不育鸡模型,以解决上述现有技术存在的问题,通过外源转入HSV-TK基因构建转基因鸡,并且在更昔洛韦辅助下特异消除鸡胚中的生殖细胞,进而可作为原始生殖细胞移植的受体鸡胚,进而实现提高移植外源原始生殖细胞的生殖系遗传效率。
为实现上述目的,本发明提供了如下方案:
本发明提供一种转基因不育鸡胚的制备方法,包括以下步骤:
(1)使用CRISPR/cas9介导的同源重组修复将HSV-TK基因定点插入到鸡原始生殖细胞特异性表达DAZL基因最后一个编码外显子的3’端,使得所述原始生殖细胞特异表达所述HSV-TK基因,回注受体鸡胚中,制备HSV-TK嵌合体鸡;
(2)经HSV-TK嵌合体鸡配种之后得到HSV-TK转基因受体鸡胚,给予更昔洛韦消除内源生殖细胞,从而获得转基因不育鸡胚。
优选的是,(1)中,将HSV-TK基因定点插入到鸡原始生殖细胞,具体包括以下步骤:
将Cas9、sgRNA质粒和供体质粒转染至原始生殖细胞,转染1周后,筛选阳性的原始生殖细胞,再接种于MKO培养基中进行扩增培养并建系,得到鸡原始生殖细胞的细胞系;其中,所述sgRNA的核苷酸序列如SEQ ID NO:1所示;所述供体质粒是将外源基因HSV-TK转入表达载体构建而成,所述HSV-TK基因的核苷酸序列如SEQ ID NO:2所示。
SgRNA(SEQ ID NO:1):agggcgcatcacttcagaaaagg。
HSV基因(SEQ ID NO:2):
ATGGCCTCGTACCCCGGCCATCAACACGCGTCTGCGTTCGACCAGGCTGCGCGTTCTCGCGGCCATAG
CAACCGACGTACGGCGTTGCGCCCTCGCCGGCAGCAAGAAGCCACGGAAGTCCGCCCGGAGCAGAA
AATGCCCACGCTACTGCGGGTTTATATAGACGGTCCCCACGGGATGGGGAAAACCACCACCACGCAA
CTGCTGGTGGCCCTGGGTTCGCGCGACGATATCGTCTACGTACCCGAGCCGATGACTTACTGGCGGGT
GCTGGGGGCTTCCGAGACAATCGCGAACATCTACACCACACAACACCGCCTCGACCAGGGTGAGATA
TCGGCCGGGGACGCGGCGGTGGTAATGACAAGCGCCCAGATAACAATGGGCATGCCTTATGCCGTGA
CCGACGCCGTTCTGGCTCCTCATATCGGGGGGGAGGCTGGGAGCTCACATGCCCCGCCCCCGGCCCT
CACCCTCATCTTCGACCGCCATCCCATCGCCGCCCTCCTGTGCTACCCGGCCGCGCGGTACCTTATGGG
CAGCATGACCCCCCAGGCCGTGCTGGCGTTCGTGGCCCTCATCCCGCCGACCTTGCCCGGCACCAAC
ATCGTGCTTGGGGCCCTTCCGGAGGACAGACACATCGACCGCCTGGCCAAACGCCAGCGCCCCGGCG
AGCGGCTGGACCTGGCTATGCTGGCTGCGATTCGCCGCGTTTACGGGCTACTTGCCAATACGGTGCGG
TATCTGCAGTGCGGCGGGTCGTGGCGGGAGGACTGGGGACAGCTTTCGGGGACGGCCGTGCCGCCC
CAGGGTGCCGAGCCCCAGAGCAACGCGGGCCCACGACCCCATATCGGGGACACGTTATTTACCCTGT
TTCGGGCCCCCGAGTTGCTGGCCCCCAACGGCGACCTGTATAACGTGTTTGCCTGGGCCTTGGACGTC
TTGGCCAAACGCCTCCGTTCCATGCACGTCTTTATCCTGGATTACGACCAATCGCCCGCCGGCTGCCG
GGACGCCCTGCTGCAACTTACCTCCGGGATGGTCCAGACCCACGTCACCACCCCCGGCTCCATACCGACGATATGCGACCTGGCGCGCACGTTTGCCCGGGAGATGGGGAGGCTAAC。
优选的是,将阳性的原始生殖细胞注射到15期鸡胚,孵化获得F0嵌合体,性成熟后通过测交获得F1代转基因不育鸡。
优选的是,所述原始生殖细胞的制备方法,包括以下步骤:
S1:制备饲养层:将MEF1代细胞经洗涤、胰酶消化后,按照MEF1代细胞和饲养层的细胞培养液的体积比为1:3传代培养,传至饲养层第三代时,用丝裂霉素处理,弃去培养液,清洗细胞后,换新的所述细胞培养液,制得饲养层;
S2:受精后的鸡蛋恒温孵化,再获取孵化后的鸡蛋的性腺,将所述性腺经洗涤、胰蛋白酶消化后,获取性腺细胞;
S3:将所述性腺细胞进行差异贴壁培养后,离心弃去上清液,重悬沉淀,然后转移到所述饲养层培养,获取所述原始生殖细胞。
优选的是,S1中,洗涤液为PBS溶液;所述胰酶的质量分数为0.05%;所述传代培养的条件为37℃、5%CO2;按照所述饲养层的细胞培养液计,所述丝裂霉素添加的质量体积分数为1%。
优选的是,S2中,所述恒温孵化的条件为:孵化温度为37.8℃,湿度60%;洗涤液为PBS溶液。
优选的是,S3中,所述重悬为用细胞培养液重悬,所述细胞培养液为DMEM/F12培养基;所述培养的条件为培养温度37.8℃、湿度60%。
优选的是,(2)中,所述更昔洛韦的浓度体外实验10μM,体内实验0.165mM。
本发明还提供了所述的制备方法制备的转基因不育鸡胚作为原始生殖细胞移植的受体鸡胚在培育转基因鸡中的应用。
本发明公开了以下技术效果:
本发明首次在禽类PGCs细胞中证明HSV-TK基因催化更昔洛韦底物磷酸化之后的细胞毒性,验证了HSV-1-TK自杀基因系统在家禽上的应用可行性。
本发明通过使用CRISPR/cas9介导的同源重组修复将HSV-TK基因定点插入到鸡原始生殖细胞特异性表达DAZL基因最后一个编码外显子的3’端,制备HSV-TK转基因鸡,在更昔洛韦辅助下特异消除鸡胚中的生殖细胞,进而可作为PGC移植的受体鸡胚。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为本发明制备生殖细胞特异性表达HSV-TK的转基因不育鸡的技术路线图;
图2为表达载体质粒18T-HSV-TK-EGFP供体质粒的构建示意图;
图3为DAZL sgRNA质粒构建示意图;
图4为体外PGCS细胞转染结果;
图5为DAZL基因的靶位点两侧进行PCR鉴定电泳图;图中,Maker为标准DNA分子,WT-L为野生型左边同源臂鉴定;HSV-L1为1号阳性HSV细胞左侧同源臂鉴定;HSV-L2为2号阳性HSV细胞左侧同源臂鉴定;WT-R为野生型右边同源臂鉴定;HSV-R1为2号阳性HSV细胞右侧同源臂鉴定;HSV-R2为2号阳性HSV细胞右侧同源臂鉴定;
图6为DAZL基因的靶位点两侧序列鉴定结果;
图7为更昔洛韦消除生殖细胞的显微镜观察结果;DAZL-HAV-TK-EGFP PGCs在10uM的更昔洛韦诱导下的凋亡;
图8为DAZL-HSV-TK-EGFP杂合子鸡胚的绿色荧光结果;
图9为出壳杂合子小鸡PCR鉴定结果;M为标准DNA分子;neg为阴性对照,A为阳性PGCs;3为杂合子;5为杂合子;27为杂合子;33为杂合子;4为杂合子;165为野生型;73为杂合子;
图10为出壳F1代转基因鸡的图片;
图11为DAZL-HSV-TK-EGFP胚胎对更昔洛韦复合物消除结果,WT为阴性对照,DAZL-HSV-TK-EGFP胚胎为阳性对照;DAZL-HSV-TK-EGFP+treated为阳性加药组;图中,箭头表示性腺中EGFP+细胞;“*”表示下层中肾的自身荧光;
图12为纯合子转基因不育鸡图以及成年公鸡及母鸡腹腔解剖图。
具体实施方式
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值,以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本发明说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等,均为开放性的用语,即意指包含但不限于。
本发明主要的技术思路,就是使用CRISPR/cas9介导的同源重组修复将HSV-TK基因定点插入到鸡原始生殖细胞特异性表达DAZL基因最后一个编码外显子的3’端,使得生殖细胞特异表达HSV-TK,制备转基因鸡,而后在鸡胚阶段给与更昔洛韦消除生殖细胞,即可作为PGC移植的受体鸡胚。技术路线图见图1。
以下是实施例方式,对上述技术思路进行进一步说明。
实施例1一种生殖细胞特异性表达HSV-TK的转基因不育鸡的制备方法
1、表达载体质粒18T-HSV-TK-EGFP供体质粒的构建
参照GeneBank中HSV-TK基因的cDNA序列,加上酶切位点后化学合成HSV-TK基因的cDNA序列(1128bp,如图2所示),将HSV-TK基因无缝插入已存在质粒DAZL左臂-2A\P2A-EGFP\DAZL右臂靶向18T载体中,进行测序验证。
2、打靶鸡生殖细胞特异性标志基因DAZL sgRNA的设计
在NCBI线上数据库中查询鸡DAZL基因(Gene ID:374054),下载基因的打靶供体质粒载体构建:使用HindIII和EcoRI双酶切pMD详细序列,利用在线基因编辑位点设计网http://chopchop.cbu.uib.no/在DAZL基因上设计编辑位点。针对DAZL基因最后一个编码外显子的3’端插入位点设计sgRNA。通过华大基因合成sgRNA序列。
DAZL sgRNA质粒构建(如图3所示):设计并合成sgRNA的Oligo DNA→退火形成双链→用限制性内切酶BbSI对px458 2.0spcas9-EGFP载体质粒进行酶切→酶切后的质粒与退火所得双链进行连接→获得打靶质粒。具体操作步骤如下:
a.反应体系:根据以下体系配制退火体系(表1):
表1
b.退火:配制完成后,涡旋5s混匀,离心30s,放入PCR仪中按照以下程序进行退火(表2):
表2
c.酶切质粒:用BbSI限制性内切酶对px458载体质粒进行酶切,涡旋混匀后,37℃,酶切2h之后进行DNA凝胶电泳,回收目的条带。
酶切质粒按照以下体系(表3)加入PCR小管中,37℃,反应2h。
表3
d.infusion连接
连接体系(表4):
表4
50℃,连接1h。
3、体外PGC细胞培养
(1)实验前准备:饲养层(MEF)的细胞培养液(DMEM/F12培养基),PBS溶液,PGC培养液以及0.05%的胰酶和0.25%的胰酶。
DMEM/F12培养液:10%FBS+90%DMEM/F12,过滤除菌后4℃备用。
(2)MEF的准备:解冻实验室原有MEF1代细胞,将长满100mm细胞培养皿的细胞加入1mL PBS溶液洗涤2次,再用0.05%的胰酶1mL摇晃至胰酶均匀接触整个培养皿消化2-3min,按照1:3的比例将细胞传代至3个100mm细胞培养皿中,放入37℃,5%的CO2细胞培养箱中培养;传至MEF第3代时,向培养液中加入1%的丝裂霉素处理,2h后弃去培养液,用PBS清洗3遍并传代至6孔板或24孔板,12h后换液。
(3)将受精后的东兰乌鸡蛋放入恒温孵化箱中,培养温度37.8℃、湿度60%,暂时不孵化的鸡胚暂时置于16℃恒温箱中。
(4)将孵化的鸡蛋取出,用弯镊从鸡胚的气室开口,夹住鸡胚头部捞出置细胞皿上。
(5)切掉头部,将鸡胚腹部朝上,用镊子轻轻划开腹部皮肤和肌肉,打开腹腔,将内脏等脏器拨开,暴露位于肾组织上的性腺。
(6)在显微镜下,用尖镊轻轻剥离整个性腺,将其在PBS中洗涤两次,洗去血液和外围的结缔组织。
(7)将性腺转移至装有20μL胰蛋白酶的EP管中,并置于37℃水浴中8分钟。
(8)用300μL CKO培养液终止消化,用移液枪轻轻吹打使细胞分散,并转移到24孔板上,用100μL CKO培养液(3对性腺/孔)洗涤EP管,终体积400μL,然后将24孔板放置细胞培养箱中进行差异贴壁5h。
CKO培养液:66%KO-DMEM+20%BRL条件培养液+7.5%FBS+2.5%Chicken Serum+4ng/mL human recombinant FGF+1X GS nucleoside supplement+0.0.1mMβ-巯基乙醇+2nM GlutaMAX+1X Anti-anti+1X non-essential amino acids(NEAA),过滤除菌后4℃备用。
(9)将培养液转移至离心管中,800rpm离心7min。弃去上清,获取PGC细胞沉淀,加入PGC细胞MKO培养液重悬,将PGC细胞移饲养层细胞上培养。
MKO培养液:0.2%Chicken Serum+2nM GlutaMAX+1X non-essential aminoacids(NEAA)+0.1mMβ-巯基乙醇+4ng/mL human recombinant FGF+sodium pyruvate:1.2mM+1X GS nucleoside supplement+1%Fetal bovine serum(Hyclone)+sodiumheparin(Sigma):100mg/mL+Activin A(PeproTech):25ng/mL+1X B27 supplement,加KO-DMEM溶液定容至100mL,过滤除菌后使用。
4、体外PGC细胞转染
按照制造商提供的说明书,使用Lipofectamine 3000(Thermo Fisher,L3000015,USA)将Cas9、sgRNA质粒和供体质粒转染至PGCs。转染1周后,用FACS分选出EGFP阳性的PGCs,接种于mKO培养基中进行扩增培养并建系。
结果图4所示,其为流式分选绿色荧光后建系的DAZL-HSV-TK-EGFP PGCs细胞。
5、DAZL-HSV-TK-EGFP PGCs细胞的鉴定
提取分选获得的绿色荧光PGC的基因组DNA,设计靶位点左右两端的引物(见表1)进行HSV-TK片段的插入鉴定,检测是否成功插入Dazl基因座中。PCR结果显示,DAZL-HSV-TK-EGFP PGCs细胞靶位点左右两端都有清晰的条带(图5),而野生型细胞的PCR泳道没有目的产物的扩增。将两端扩增产物回收并测序,发现与预测的DAZL-HSV-TK-EGFP片段一致(图6),说明获得的绿色荧光PGC细胞成功插入HSV-TK-EGFP基因。
PGC鉴定PCR引物见表5:
表5
PCR反应体系及条件2×Taq PCR预混试剂Ⅱ(KT211)(天根),见表5:
表6
PCR反应程序按照天根2xTaq PCR预混试剂II(KT211)说明书进行,见表7:
表7
PCR反应产物用1.5%的琼脂糖凝胶电泳检测。110V,20min。
结果如图5所示,其为靶位点两端的PCR鉴定结果;图6为两端PCR产物的测序结果。
6、HSV-TK系统特异性诱导PGCs的消除
为了鉴定HSV-TK是否能特异性诱导PGC细胞凋亡,在24孔板上接种WT PGC和DAZL-HSV-TK-EGFP PGCs,选用10μM的更昔洛韦底物分别处理WT PGC与DAZL-HSV-TK-EGFP PGCs细胞系,培养48h后,在显微镜下进行观察,拍照。
结果如图7所示,阴性对照加药无影响,阳性对照显示Bright和绿光,阳性加药组显示阳性PGC在10μM药物诱导下发生凋亡。
7、生殖嵌合体鸡的制备
(1)将63枚已受精的广西麻鸡种蛋置恒温孵化箱中,在37.8℃,湿度60%条件下孵育53h。
(2)将构建的DAZL-HSV-TK-EGFP PGCs细胞收集到离心管中,700rpm,离心5分钟。离心后用1mL PGC培养液重悬,进行细胞计数,计算细胞浓度,稀释至105个/mL,加入445μL的已过滤除菌的台盼蓝染色液和4mL细胞悬液混匀。吸取100μL做液滴,做40个。在细胞皿中添加矿物油没过液滴表面。静置几分钟后,将台盼蓝液滴中的细胞以一个方向轻轻晃动,使细胞聚集在液滴的中心,在显微镜下观察细胞状态,死细胞被台盼蓝染色,活细胞无色。
(3)显微注射
a:鸡胚开口:用75%的酒精擦拭孵化53h的鸡胚进行消毒,标注好气室,用弯镊在气室轻轻打开指甲盖大小的孔,观察到红色跳动心脏。再用移液枪加入200μL含双抗的PBS,于体式显微镜下用细镊子缓慢撕开内壳膜,暴露出心脏。
b:吸细胞:将注射针插入蓝色液滴中,吸取液滴中的无色细胞到注射针内,将注射针内的液体体积控制在2μL左右。
c:细胞注射:在显微镜下找到发育的鸡胚心脏,将注射针内细胞液体缓慢注射到发育53h的心脏处,含有细胞的蓝色液体随心脏泵入鸡胚,注射针内保留一点液体时轻轻拔出,以防止空气注入。
d:封口:在酒精灯上加热钥匙,用热的钥匙融化封口膜边缘使其与鸡蛋表面紧密相贴,以防空气渗入。
(4)鸡胚孵化:将注射完的受体鸡胚放回到孵化箱中继续孵化至小鸡破壳。
8、利用生殖嵌合体鸡配种罗曼粉母鸡制备DAZL-HSV-TK-EGFP杂合子:鸡蛋孵化E6.5天鸡胚进行解剖,分离性腺,观察到绿色荧光PGC情况,这表明本实验成功获得DAZL-HSV-TK-EGFP杂合子(如图8所示)。
出壳小鸡(如图10所示)同上述条件按照天根2xTaq PCR预混试剂II(KT211)说明书进行PCR鉴定,鉴定引物为F(SEQ ID NO:7):5'-GTTCTCGCGGCCATAGCAA-3';R(SEQ ID NO:8):5'-ATATGAGGAGCCAGAACGGC-3'。
结果如图9所示,PCR出现阳性条带为杂合子小鸡。
9、利用DAZL-HSV-TK-EGFP杂合子自交获得纯合子,并验证体内生殖细胞的消除。16期HH(第2.5天)胚胎顶部添加100μL PBS(含2%青霉素/链霉素和0.165mM浓度GCV化合物)。
10、更昔洛韦处理,并孵育至胚胎发育的第6.5天。结果如图11所示,WT野生型不发绿光为阴性对照,DAZL-HSV-TK-EGFP不加药为阳性对照,DAZL-HSV-TK-EGFP加药后绿光消失。
待成年后公鸡无精液,母鸡不产蛋,成功获得转基因不育公鸡和母鸡,分别解剖腹腔观察睾丸和卵巢形态,(如图12所示)。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (9)
1.一种转基因不育鸡胚的制备方法,其特征在于,包括以下步骤:
(1)使用CRISPR/cas9介导的同源重组修复将HSV-TK基因定点插入到鸡原始生殖细胞特异性表达DAZL基因最后一个编码外显子的3’端,使得所述原始生殖细胞特异表达所述HSV-TK基因,回注受体鸡胚中,制备HSV-TK嵌合体鸡;
(2)经HSV-TK嵌合体鸡配种之后得到HSV-TK转基因受体鸡胚,给予更昔洛韦消除内源生殖细胞,从而获得转基因不育鸡胚。
2.如权利要求1所述的制备方法,其特征在于,(1)中,将HSV-TK基因定点插入到鸡原始生殖细胞,具体包括以下步骤:
将Cas9、sgRNA质粒和供体质粒转染至原始生殖细胞,转染3d后,筛选阳性的原始生殖细胞,再接种于MKO培养基中进行扩增培养并建系,得到鸡原始生殖细胞的细胞系;其中,所述sgRNA的核苷酸序列如SEQ ID NO:1所示;所述供体质粒是将外源基因HSV-TK转入表达载体构建而成,所述HSV-TK基因的核苷酸序列如SEQ ID NO:2所示。
3.如权利要求2所述的制备方法,其特征在于,将阳性的原始生殖细胞注射到15期鸡胚,孵化获得F0嵌合体,性成熟后通过测交获得F1代转基因不育鸡。
4.如权利要求1所述的制备方法,其特征在于,所述原始生殖细胞的制备方法,包括以下步骤:
S1:制备饲养层:将MEF1代细胞经洗涤、胰酶消化后,按照MEF1代细胞和饲养层的细胞培养液的体积比为1:3传代培养,传至饲养层第三代时,用丝裂霉素处理,弃去培养液,清洗细胞后,换新的所述细胞培养液,制得饲养层;
S2:受精后的鸡蛋恒温孵化,再获取孵化后的鸡蛋的性腺,将所述性腺经洗涤、胰蛋白酶消化后,获取性腺细胞;
S3:将所述性腺细胞进行差异贴壁培养后,离心弃去上清液,重悬沉淀,然后转移到所述饲养层培养,获取所述原始生殖细胞。
5.如权利要求4所述的制备方法,其特征在于,S1中,洗涤液为PBS溶液;所述胰酶的质量分数为0.05%;所述传代培养的条件为37℃、5%CO2;按照所述饲养层的细胞培养液计,所述丝裂霉素添加的质量体积分数为1%。
6.如权利要求4所述的制备方法,其特征在于,S2中,所述恒温孵化的条件为:孵化温度为37.8℃,湿度60%;洗涤液为PBS溶液。
7.如权利要求4所述的制备方法,其特征在于,S3中,所述重悬为用细胞培养液重悬,所述细胞培养液为DMEM/F12培养基;所述培养的条件为培养温度37.8℃、湿度60%。
8.如权利要求1所述的制备方法,其特征在于,(2)中,所述更昔洛韦的浓度体外实验10μM,体内实验0.165mM。
9.如权利要求1-8任一项所述的制备方法制备的转基因不育鸡胚作为原始生殖细胞移植的受体鸡胚在培育转基因鸡中的应用。
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