CN114015705A - 一种小鼠体外受精繁育性别选择方法 - Google Patents
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Abstract
本发明涉及一种小鼠体外受精繁育性别选择方法,利用基因编辑技术,在雄性小鼠Y染色体上插入luciferase基因,通过胚胎注射和移植,通过基因鉴定得到luc‑Y雄性小鼠;luc‑Y雄性小鼠的精子保存液中加入luciferase的底物,luc‑Y精子发出波长560nm的荧光,正常X精子无荧光,通过流式细胞分选仪分选出luc‑Y精子和正常X精子,使用荧光显微镜观察分选后的luc‑Y精子和正常X精子,通过荧光判断正常X精子中luc‑Y精子的比例,若比例超标则将正常X精子重新放入流式细胞分选仪进行分选,直至正常X精子纯度达标为止。本发明可以方便且直观的通过肉眼判断流式分选的结果,有效提高小鼠X精子和Y精子的筛选精度。
Description
技术领域:
本发明涉及生物实验技术领域,具体涉及一种小鼠体外受精繁育性别选择方法。
背景技术:
小鼠体外受精用于净化和扩繁。一般自然受精会得到雌雄各半的子代。但是不同的实验对动物的性别有选择性的需求。例如大规模扩繁需要数量更多的雌性小鼠,而一些动物模型则需要雄性小鼠来做实验。因此,为了得到目的数量特定性别的小鼠,通过小鼠精子和卵细胞自然结合会导致饲养工作量和费用翻倍,浪费人力物力,并且处死不需要的性别的小鼠也有违动物伦理。因此我们开发一种在小鼠体外受精过程中可以选择性别的方法,以便快速高效的得到想要的性别的小鼠。专利号为ZL201711338448.4的专利提供了一种小鼠X/Y精子分离精液的生产方法及应用,它是应用小鼠X、Y精子DNA含量的差异,通过流式细胞方式分离X和Y精子,然后制成性别控制精液,用于小鼠的体外受精、人工受精和胞质内精子注射,按照需要定向繁育雄鼠或雌鼠,或者通过小鼠X/Y精子分离精液生产性控胚胎,再进行胚胎移植,加快特殊品系小鼠的繁育速度。通过添加上机分离液来保护小鼠的鲜精,有效的保护了小鼠精子的活力,提高分离效率。但是这种方法是直接使用流式细胞分选仪对小鼠的精液进行流式分选以分离X精子和Y精子,且设定分选标准为DNA含量相差3.2%,可选区间较小,分选得到的精子容易发生混杂,加上由于流式细胞分选仪始终存在筛选误差,并且分选结果没有简便的检测精子纯度的质控方法,筛选精度有限。
发明内容:
(一)解决的技术问题
本发明旨在提供一种小鼠体外受精繁育性别选择方法,解决现有技术直接使用流式细胞分选仪对小鼠的精液进行流式分选以分离X精子和Y精子,可选区间较小,分选得到的精子容易发生混杂,没有简便的检测精子纯度的质控方法,筛选精度有限的问题。
(二)技术方案
为解决上述技术问题,本发明采用如下技术方案:一种小鼠体外受精繁育性别选择方法,包括以下步骤:
(1)利用基因编辑技术,在雄性小鼠Y染色体上插入luciferase基因,通过胚胎注射和移植,通过基因鉴定得到luc-Y雄性小鼠;
(2)取luc-Y雄性小鼠的精子,包括luc-Y精子和正常X精子,在精子保存液中加入luciferase底物,luc-Y精子发出波长560nm的荧光,正常X精子无荧光,通过流式细胞分选仪分选出luc-Y精子和正常X精子;
(3)取分选出的luc-Y精子和正常X精子各40-50万个,存放于含有luciferase底物的保存液中,使用电子显微镜观察分离后的luc-Y精子和正常X精子,通过荧光判断正常X精子中luc-Y精子的比例,若比例超标则将正常X精子重新放入流式细胞分选仪进行分选,直至正常X精子纯度达标为止;
(4)将luc-Y精子和正常卵细胞体外受精,将受精卵移植到代孕母鼠体内,代孕母鼠生出的子代将会全部都是雄性子代;将正常X精子和正常卵细胞体外受精,将受精卵移植到代孕母鼠体内,代孕母鼠生出的子代将会全部都是雌性子代。
进一步地,所述luc-Y雄性小鼠获得方法如下:
(1)通过体外表达纯化制备得到具有核酸酶活性的Cas9蛋白备用;
(2)针对小鼠Y染色体上的基因间区设计sgRNA,并进行软件分析以确保所述sgRNA没有预测的脱靶结合位点,采用转录试剂盒,体外转录所述sgRNA,转录好的所述sgRNA备用;
(3)根据打靶位点,选取打靶位点上游1000bp序列作为左同源臂,选取打靶位点下游1000bp序列作为右同源臂,进行载体构建,原件包括左同源臂、启动子CMV、luciferaseCDS、polyA、右同源臂,以单链DNA作为打靶载体备用;
(4)将所述Cas9蛋白、所述sgRNA及所述单链DNA打靶载体混合好,进行胚胎注射及移植;
(5)移植后出生的小鼠标记为F0代,取F0代雄性小鼠进行基因鉴定,在小鼠Y染色体基因组上,在打靶位点上游设计上游引物,在luciferase CDS序列上设计下游引物,引物设计时目的条带在500-1000bp,进行PCR鉴定,有条带的为阳性luc-Y小鼠,无条带的则为阴性野生型小鼠。
进一步地,所述luc-Y精子和所述X精子的分选方法如下:
(1)将性成熟的luc-Y雄鼠颈椎脱臼处死,将输精管和附睾放入精子保存液中,用镊子轻轻将精子挤出来,当精子充分游离出输精管附睾后,将输精管和附睾组织取出,用移液枪把精子和保存液充分混合均匀并转移至5ml离心管中;
(2)根据荧光波长设置流式细胞分选参数,荧光波长560nm,细胞流速100-200个/秒,选取细胞40-50万个,上机前向精子保存液中加入luciferase底物,并迅速上机进行分选,分选过程中适当添加luciferase底物,以保证有清晰稳定的荧光信号;
(3)根据荧光强度圈出阳性和阴性区域,只取荧光最强和最弱的部分,舍弃中间荧光较弱的部分,防止荧光背景的干扰,增大可信度,分别收集阳性细胞和阴性细胞,将细胞收集在TYH培养基中使其获能,阳性细胞即为luc-Y精子,阴性细胞即为X精子。
雄性小鼠的繁育过程如下:
(1)收集卵母细胞,处死超排后的雌鼠,取出两侧输卵管,置入培养皿取出卵丘细胞团放入TYH培养基中;
(2)将分选得到的luc-Y精子滴加到卵子液滴中,将精卵混合后置于培养箱中培养,以完成体外受精;
(3)体外受精完成后将胚胎移植入假孕母鼠子宫,21天后出生小鼠即为雄性小鼠。
雌性小鼠的繁育过程如下:
(1)收集卵母细胞,处死超排后的雌鼠,取出两侧输卵管,置入培养皿取出卵丘细胞团放入TYH培养基中;
(2)将分选得到的X精子滴加到卵子液滴中,将精卵混合后置于培养箱中培养,以完成体外受精;
(3)体外受精完成后将胚胎移植入假孕母鼠子宫,21天后出生小鼠即为雌性小鼠。
(三)有益效果
相对于现有技术,本发明产生的有益效果是:利用基因编辑技术,在雄性小鼠Y染色体上的基因间区插入luciferase基因,得到luc-Y雄性小鼠,然后对luc-Y雄性小鼠的精子进行流式分选,通过luciferase蛋白能催化底物发出荧光的功能,区分正常X精子和luc-Y精子,以辅助进行流式分选。分选标准是有或无荧光,并且可以舍弃中间荧光较弱的部分,只取荧光最强和最弱的部分,防止荧光背景的干扰,增大可信度;并且分选后的精子可以通过荧光显微镜直接观察分选结果,在不影响小鼠本身生长发育的前提下,可以有效提高性别筛选精度,避免无用性别小鼠的浪费。
附图说明:
为了更清楚地说明本发明实施例中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍。
图1是本发明实施例所述基因编辑原理图;
图2是本发明实施例所述流式分选原理图;
图3是本发明实施例所述流式分选荧光强度与分选数量对比图;
具体实施方式:
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述。
小鼠Y染色体上没有基因表达的基因间区,在不影响正常基因表达的情况下,可以通过基因编辑技术插入luciferase基因。Luciferase基因是萤火虫基因组中编码荧光素酶的基因,由于luciferase蛋白功能单一,能催化底物发出荧光,且不是小鼠基因组中的基因,所以插入luciferase基因对小鼠的生长发育无影响。
小鼠体外受精繁育性别选择方法,包括以下步骤:
(1)利用基因编辑技术(原理见图1),在雄性小鼠Y染色体上插入luciferase基因,通过胚胎注射和移植,通过基因鉴定得到luc-Y雄性小鼠;
(2)取luc-Y雄性小鼠的精子,包括luc-Y精子和正常X精子,在精子保存液中加入luciferase底物(货号40903ES01),luc-Y精子发出波长560nm的荧光,正常X精子无荧光,通过流式细胞分选仪分选出luc-Y精子和正常X精子(流式分选原理见图2);
(3)取分选出的luc-Y精子和正常X精子各40-50万个,存放于含有luciferase底物的保存液中,使用荧光显微镜观察分选后的luc-Y精子和正常X精子,通过荧光判断正常X精子中luc-Y精子的比例,若比例超标则将正常X精子重新放入流式细胞分选仪进行分选,直至正常X精子纯度达标为止。
luc-Y雄性小鼠获得方法如下:
(1)通过体外表达纯化制备得到具有核酸酶活性的Cas9蛋白备用;
(2)针对小鼠Y染色体上的基因间区设计sgRNA,并进行软件分析以确保sgRNA没有预测的脱靶结合位点,采用转录试剂盒,体外转录sgRNA,转录好的sgRNA备用;
(3)根据打靶位点,选取打靶位点上游1000bp序列作为左同源臂,选取打靶位点下游1000bp序列作为右同源臂,进行载体构建,原件包括左同源臂、启动子CMV、luciferaseCDS、polyA、右同源臂,以单链DNA作为打靶载体备用;
(4)将Cas9蛋白、sgRNA及单链DNA打靶载体混合好,进行胚胎注射及移植;
(5)移植后出生的小鼠标记为F0代,取F0代雄性小鼠进行基因鉴定,在小鼠Y染色体基因组上,在打靶位点上游设计上游引物,在luciferase CDS序列上设计下游引物,引物设计时目的条带在500-1000bp左右为宜,进行PCR鉴定,有条带的为阳性luc-Y小鼠,无条带的则为阴性野生型小鼠。
luc-Y精子和X精子的分选方法如下:
(1)将性成熟的luc-Y雄鼠颈椎脱臼处死,将输精管和附睾放入精子保存液中,用30号针头的尖在附睾上划3~5次,并向下划开输精管用镊子轻轻将精子挤出来,当精子充分游离出输精管附睾后,在37℃的培养箱中将精子及输精管和附睾组织共培养10~15min,然后将输精管和附睾组织取出,用移液枪把精子和保存液充分混合均匀并转移至5ml离心管中;
(2)根据荧光波长设置流式细胞分选参数,荧光波长560nm,细胞流速100-200个/秒,选取细胞40-50万个,上机前向精子保存液中加入luciferase底物,并迅速上机进行分选,分选过程中适当添加luciferase底物,以保证有清晰稳定的荧光信号;
(3)根据荧光强度圈出阳性和阴性区域,只取荧光最强和最弱的部分,舍弃中间荧光较弱的部分,防止荧光背景的干扰,增大可信度,分别收集阳性细胞和阴性细胞,将细胞收集在TYH培养基中使其获能,阳性细胞即为luc-Y精子,阴性细胞即为X精子。
雄性小鼠的繁育过程如下:
(1)收集卵母细胞,处死超排后的雌鼠,取出两侧输卵管,置入培养皿取出卵丘细胞团放入TYH培养基中;
(2)将分选得到的luc-Y精子滴加到卵子液滴中,将精卵混合后置于培养箱中培养,以完成体外受精;
(3)体外受精完成后将胚胎移植入假孕母鼠子宫,21天后出生小鼠即为雄性小鼠。
雌性小鼠的繁育过程如下:
(1)收集卵母细胞,处死超排后的雌鼠,取出两侧输卵管,置入培养皿取出卵丘细胞团放入TYH培养基中;
(2)将分选得到的X精子滴加到卵子液滴中,将精卵混合后置于培养箱中培养,以完成体外受精;
(3)体外受精完成后将胚胎移植入假孕母鼠子宫,21天后出生小鼠即为雌性小鼠。
综上所述,本发明提供的小鼠体外受精繁育性别选择方法,解决现有技术直接使用流式细胞分选仪对小鼠的精液进行流式分选以分离X精子和Y精子,可选区间较小,分选得到的精子容易发生混杂,没有简便的检测精子纯度的质控方法,筛选精度有限的问题。
上面以举例方式对本发明进行了说明,但本发明不限于上述具体实施例,凡基于本发明所做的任何改动或变型均属于本发明要求保护的范围。
Claims (3)
1.一种小鼠体外受精繁育性别选择方法,其特征在于,包括以下步骤:
(1)利用基因编辑技术,在雄性小鼠Y染色体上的基因间区插入luciferase基因,通过胚胎注射和移植,通过基因鉴定得到luc-Y雄性小鼠;
(2)取luc-Y雄性小鼠的精子,包括luc-Y精子和正常X精子,在精子保存液中加入luciferase底物,luc-Y精子发出波长560nm的荧光,正常X精子无荧光,通过流式细胞分选仪分选出luc-Y精子和正常X精子;
(3)取分选出的luc-Y精子和正常X精子各40-50万个,存放于含有luciferase底物的保存液中,使用荧光显微镜观察分离后的luc-Y精子和正常X精子,通过荧光判断正常X精子中luc-Y精子的比例,若比例超标则将正常X精子重新放入流式细胞分选仪进行分选,直至正常X精子纯度达标为止;
(4)将luc-Y精子和正常卵细胞体外受精,将受精卵移植到代孕母鼠体内,代孕母鼠生出的子代将会全部都是雄性子代;将正常X精子和正常卵细胞体外受精,将受精卵移植到代孕母鼠体内,代孕母鼠生出的子代将会全部都是雌性子代。
2.根据权利要求1所述的一种小鼠体外受精繁育性别选择方法,其特征在于,所述luc-Y雄性小鼠获得方法如下:
(1)通过体外表达纯化制备得到具有核酸酶活性的Cas9蛋白备用;
(2)针对小鼠Y染色体上的基因间区设计sgRNA,并进行软件分析以确保所述sgRNA没有预测的脱靶结合位点,采用转录试剂盒,体外转录所述sgRNA,转录好的所述sgRNA备用;
(3)根据打靶位点,选取打靶位点上游1000bp序列作为左同源臂,选取打靶位点下游1000bp序列作为右同源臂,进行载体构建,原件包括左同源臂、启动子CMV、luciferaseCDS、polyA、右同源臂,以单链DNA作为打靶载体备用;
(4)将所述Cas9蛋白、所述sgRNA及所述单链DNA打靶载体混合好,进行胚胎注射及移植;
(5)移植后出生的小鼠标记为F0代,取F0代雄性小鼠进行基因鉴定,在小鼠Y染色体基因组上,在打靶位点上游设计上游引物,在luciferase CDS序列上设计下游引物,引物设计时目的条带在500-1000bp,进行PCR鉴定,有条带的为阳性luc-Y小鼠,无条带的则为阴性野生型小鼠。
3.根据权利要求1所述的一种小鼠体外受精繁育性别选择方法,其特征在于,所述luc-Y精子和所述X精子的分选方法如下:
(1)将性成熟的luc-Y雄鼠颈椎脱臼处死,将输精管和附睾放入精子保存液中,用镊子轻轻将精子挤出来,当精子充分游离出输精管附睾后,将输精管和附睾组织取出,用移液枪把精子和保存液充分混合均匀并转移至5ml离心管中;
(2)根据荧光波长设置流式细胞分选参数,荧光波长560nm,细胞流速100-200个/秒,选取细胞40-50万个,上机前向精子保存液中加入luciferase底物,并迅速上机进行分选,分选过程中适当添加luciferase底物,以保证有清晰稳定的荧光信号;
(3)根据荧光强度圈出阳性和阴性区域,只取荧光最强和最弱的部分,舍弃中间荧光较弱的部分,防止荧光背景的干扰,增大可信度,分别收集阳性细胞和阴性细胞,将细胞收集在TYH培养基中使其获能,阳性细胞即为luc-Y精子,阴性细胞即为X精子。
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Citations (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1990013303A1 (en) * | 1989-05-10 | 1990-11-15 | The United States Of America, Represented By The Secretary, United States Department Of Commerce | Method to preselect the sex of offspring |
GB9926161D0 (en) * | 1999-11-04 | 2000-01-12 | Pig Improvement Company Uk Lim | Methods |
ITMI20000030A0 (it) * | 2000-01-14 | 2000-01-14 | Istituto Sperimentale Italiano | Procedimento per la produzione di embrioni non umani di sesso predeterminato ad alto valore genetico |
WO2001047353A1 (en) * | 1999-12-27 | 2001-07-05 | Chengyu Liu | Controlling offspring's sex ratio by targeting transgenes onto the sex chromosomes |
WO2004104178A2 (en) * | 2003-05-15 | 2004-12-02 | Xy, Inc. | Efficient haploid cell sorting for flow cytometer systems |
CN1745626A (zh) * | 2004-09-06 | 2006-03-15 | 戴蕴平 | 一种经修饰的雄性哺乳动物体细胞系及其制备方法与应用 |
US20060064770A1 (en) * | 2004-09-20 | 2006-03-23 | David Frendewey | Methods for identifying a cell or an embryo carrying a Y chromosome |
CN104388560A (zh) * | 2014-11-14 | 2015-03-04 | 中国农业大学 | 一种y染色体标记方法及其应用 |
CN105658050A (zh) * | 2013-05-31 | 2016-06-08 | 重组股份有限公司 | 用于制备具有可分选精子的动物的遗传技术 |
CA2998642A1 (en) * | 2015-09-17 | 2017-03-23 | Regeneron Pharmaceuticals, Inc. | Selection of pluripotent cells for production of fertile xy female mice |
WO2018013759A1 (en) * | 2016-07-13 | 2018-01-18 | Yorktown Technologies, L.P. | Poultry and offspring sex selection |
CN108103012A (zh) * | 2017-12-14 | 2018-06-01 | 内蒙古赛科星家畜种业与繁育生物技术研究院有限公司 | 小鼠x/y精子分离精液的生产方法及应用 |
CN109402244A (zh) * | 2018-12-20 | 2019-03-01 | 广西大学 | 一种哺乳动物胚胎性别鉴定方法 |
CA3082620A1 (en) * | 2017-12-04 | 2019-06-13 | Chromoxyion Pty Ltd | Materials and methods including for sex selection |
CN111549070A (zh) * | 2020-04-26 | 2020-08-18 | 华南农业大学 | 对x染色体多拷贝基因进行编辑实现动物性别控制的方法 |
CN112768076A (zh) * | 2021-02-01 | 2021-05-07 | 华中科技大学同济医学院附属协和医院 | 一种食管癌化疗发生骨髓抑制的风险预测方法 |
-
2021
- 2021-11-28 CN CN202111426961.5A patent/CN114015705A/zh active Pending
Patent Citations (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1990013303A1 (en) * | 1989-05-10 | 1990-11-15 | The United States Of America, Represented By The Secretary, United States Department Of Commerce | Method to preselect the sex of offspring |
GB9926161D0 (en) * | 1999-11-04 | 2000-01-12 | Pig Improvement Company Uk Lim | Methods |
WO2001047353A1 (en) * | 1999-12-27 | 2001-07-05 | Chengyu Liu | Controlling offspring's sex ratio by targeting transgenes onto the sex chromosomes |
ITMI20000030A0 (it) * | 2000-01-14 | 2000-01-14 | Istituto Sperimentale Italiano | Procedimento per la produzione di embrioni non umani di sesso predeterminato ad alto valore genetico |
WO2004104178A2 (en) * | 2003-05-15 | 2004-12-02 | Xy, Inc. | Efficient haploid cell sorting for flow cytometer systems |
CN1745626A (zh) * | 2004-09-06 | 2006-03-15 | 戴蕴平 | 一种经修饰的雄性哺乳动物体细胞系及其制备方法与应用 |
US20060064770A1 (en) * | 2004-09-20 | 2006-03-23 | David Frendewey | Methods for identifying a cell or an embryo carrying a Y chromosome |
CN105658050A (zh) * | 2013-05-31 | 2016-06-08 | 重组股份有限公司 | 用于制备具有可分选精子的动物的遗传技术 |
CN104388560A (zh) * | 2014-11-14 | 2015-03-04 | 中国农业大学 | 一种y染色体标记方法及其应用 |
CA2998642A1 (en) * | 2015-09-17 | 2017-03-23 | Regeneron Pharmaceuticals, Inc. | Selection of pluripotent cells for production of fertile xy female mice |
WO2018013759A1 (en) * | 2016-07-13 | 2018-01-18 | Yorktown Technologies, L.P. | Poultry and offspring sex selection |
CA3082620A1 (en) * | 2017-12-04 | 2019-06-13 | Chromoxyion Pty Ltd | Materials and methods including for sex selection |
CN108103012A (zh) * | 2017-12-14 | 2018-06-01 | 内蒙古赛科星家畜种业与繁育生物技术研究院有限公司 | 小鼠x/y精子分离精液的生产方法及应用 |
CN109402244A (zh) * | 2018-12-20 | 2019-03-01 | 广西大学 | 一种哺乳动物胚胎性别鉴定方法 |
CN111549070A (zh) * | 2020-04-26 | 2020-08-18 | 华南农业大学 | 对x染色体多拷贝基因进行编辑实现动物性别控制的方法 |
CN112768076A (zh) * | 2021-02-01 | 2021-05-07 | 华中科技大学同济医学院附属协和医院 | 一种食管癌化疗发生骨髓抑制的风险预测方法 |
Non-Patent Citations (1)
Title |
---|
梅洁;桂建芳;: "鱼类性别异形和性别决定的遗传基础及其生物技术操控", 中国科学:生命科学 * |
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