CN104267011A - Transgenic sheep oocyte screening method - Google Patents
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Abstract
一种转基因羊卵母细胞的筛选方法，涉及一种卵母细胞的筛选方法。本发明是要解决现有的转基因羊卵母细胞质量检测方法中存在取材困难，实验周期长的问题。方法：一、对转基因羊和非转基因羊进行超数排卵，收集不同发育时期的卵母细胞；二、用多聚甲醛固定卵母细胞，依次用Triton-100、HCl、BSA封闭液处理；三、将卵母细胞经PBS吹洗，加入一抗，孵育过夜，然后于二抗中孵育，染色，制片，显微镜观察并拍照；四、采用图像分析软件将同一发育时期的转基因羊和非转基因羊的卵母细胞进行荧光强度定量分析比较，选择与非转基因羊相比无显著性差异的转基因羊卵母细胞，即完成转基因羊卵母细胞的筛选。本发明用于筛选转基因羊卵母细胞。
A method for screening transgenic sheep oocytes relates to a method for screening oocytes. The invention aims to solve the problems of difficulty in obtaining materials and long experiment period in the existing method for detecting the quality of transgenic sheep oocytes. Methods: 1. Perform superovulation on transgenic sheep and non-transgenic sheep, and collect oocytes at different developmental stages; 2. Fix oocytes with paraformaldehyde, and sequentially treat them with Triton-100, HCl, and BSA blocking solution; 3. 1. Wash oocytes with PBS, add primary antibody, incubate overnight, then incubate in secondary antibody, stain, prepare slices, observe under microscope and take pictures; 4. Use image analysis software to compare transgenic sheep and non-transgenic sheep at the same development The oocytes of the sheep were quantitatively analyzed and compared with the fluorescence intensity, and the transgenic sheep oocytes with no significant difference compared with the non-transgenic sheep were selected, that is, the screening of the transgenic sheep oocytes was completed. The invention is used for screening transgenic sheep oocytes.
Description
技术领域technical field
本发明涉及一种卵母细胞的筛选方法。The invention relates to a method for screening oocytes.
背景技术Background technique
自1982年转基因鼠问世以来，转基因动物在疾病模型、医药、品种培育、环境保护及资源保藏等领域取得了突破性的进展。随着转基因动物应用的不断扩大和人们对生物安全的担忧和关注，转基因动物生物安全研究应运而生。转基因动物生物安全研究立足于转基因动物及其产品的潜在风险，它与目的基因及其操作方式密切相关。转基因动物的目的基因和表达产物，以及在长期使用与积累过程中，都有可能带来新的风险。为防范转基因生物对环境及人类健康的风险,并促进转基因产业的健康发展,转基因动物研究的每一环节都要经过严密的检测和评价，尽可能地防范一切有害于人类健康、动物健康和环境安全的因素。Since the advent of transgenic mice in 1982, transgenic animals have made breakthroughs in the fields of disease models, medicine, breed breeding, environmental protection and resource preservation. With the continuous expansion of the application of transgenic animals and people's concerns and concerns about biosafety, research on biosafety of transgenic animals has emerged as the times require. Biosafety research on transgenic animals is based on the potential risks of transgenic animals and their products, which are closely related to the target gene and its operation method. The target gene and expression products of transgenic animals, as well as the long-term use and accumulation process, may bring new risks. In order to prevent the risks of genetically modified organisms to the environment and human health, and to promote the healthy development of the genetically modified industry, every link in the research of genetically modified animals must undergo strict testing and evaluation, and prevent as much as possible any harm to human health, animal health and the environment. safety factor.
DNA甲基化是最基本的表观遗传修饰方式之一，对哺乳动物的发育至关重要。DNA甲基化是指在DNA甲基化转移酶(Dnmts)的作用下，由S-腺苷甲硫氨酸(SAM)作为甲基供体，在基因组内CpG二核苷酸中胞嘧啶的5位碳原子加上一个甲基基团(5mC)。DNA甲基化分为维持甲基化和从头甲基两种模式，参与很多重要的发育事件，例如，基因表达调控、基因组印迹、X染色体失活及异染色质的形成。DNA甲基化对于哺乳动物生殖细胞的分化和发育至关重要，基因组DNA甲基化处于动态的重编程过程中，主要表现为原始生殖细胞要经历一个普遍的去甲基化过程，而成熟配子的基因组则是高度甲基化的。受精后，合子基因组也发生广泛的主动去甲基化，而植入后胚胎则发生普遍的新甲基化。DNA methylation is one of the most basic epigenetic modifications and is critical to mammalian development. DNA methylation refers to the removal of cytosine from CpG dinucleotides in the genome by S-adenosylmethionine (SAM) as a methyl donor under the action of DNA methyltransferase (Dnmts). The 5 carbon atom plus a methyl group (5mC). DNA methylation is divided into two modes of maintenance methylation and de novo methylation, and is involved in many important developmental events, such as regulation of gene expression, genomic imprinting, X chromosome inactivation, and heterochromatin formation. DNA methylation is crucial to the differentiation and development of mammalian germ cells. Genomic DNA methylation is in a dynamic reprogramming process, mainly manifested in that primordial germ cells undergo a general demethylation process, while mature gametes genome is highly methylated. After fertilization, there is also extensive active demethylation of the zygotic genome, whereas there is pervasive demethylation in post-implantation embryos.
通过显微注射方法生产的转基因羊，外源基因的随机插入，以及目的基因过表达，是否影响卵母细胞的甲基化水平尚不清楚。另外，显微操作也是影响卵母细胞甲基化水平的重要因素。由于转基因动物稀缺，尤其是大家畜动物，因此从生殖健康这个角度评估转基因动物安全性困难重重，如取材困难，实验周期长等。我们发明新的评估方法是通过超数排卵技术采集体内不同发育时期的卵母细胞，采用免疫荧光染色的方法，利用confocal采集图片，检测F0代转基因和非转基因羊卵母细胞全基因组甲基化水平差异，来评估转基因羊生殖健康状况。Whether transgenic sheep produced by microinjection, random insertion of exogenous genes, and overexpression of target genes affect the methylation level of oocytes is unclear. In addition, micromanipulation is also an important factor affecting the methylation level of oocytes. Due to the scarcity of transgenic animals, especially large livestock animals, it is very difficult to evaluate the safety of transgenic animals from the perspective of reproductive health, such as difficulty in obtaining materials and long experiment period. We invented a new evaluation method to collect oocytes at different developmental stages in the body through superovulation technology, use immunofluorescence staining method, use confocal to collect pictures, and detect the genome-wide methylation of F0 generation transgenic and non-transgenic sheep oocytes To evaluate the reproductive health status of transgenic sheep.
发明内容Contents of the invention
本发明是要解决现有的转基因羊卵母细胞质量检测方法中存在取材困难，实验周期长的问题，提供一种转基因羊卵母细胞的筛选方法。The invention aims to solve the problems of difficulty in obtaining materials and long experiment period in the existing method for detecting the quality of transgenic sheep oocytes, and provides a screening method for transgenic sheep oocytes.
本发明转基因羊卵母细胞的筛选方法，按以下步骤进行：The screening method of transgenic sheep oocyte of the present invention is carried out according to the following steps:
一、采用CIDR+FSH的方法对转基因羊和非转基因羊进行超数排卵，收集不同发育时期的卵母细胞；1. Using the method of CIDR+FSH to superovulate transgenic sheep and non-transgenic sheep, and collect oocytes at different developmental stages;
二、用体积百分浓度为4％的多聚甲醛固定卵母细胞至少30分钟，然后依次用体积百分浓度为1％的Triton-100处理30分钟、体积百分浓度为1％的HCl处理30分钟、2mg/mL的BSA封闭液封闭30分钟；2. Fix oocytes with 4% paraformaldehyde by volume for at least 30 minutes, and then treat them with 1% Triton-100 for 30 minutes and 1% HCl for 30 minutes. 30 minutes, 2mg/mL BSA blocking solution for 30 minutes;
三、将卵母细胞经PBS吹洗3次，然后加入一抗，4℃孵育过夜，然后于FITC标记的山羊抗小鼠二抗中37℃孵育1.5h，接着于碘化丙碇中室温染色10min，制片，Confocal显微镜观察并拍照；3. Wash oocytes with PBS 3 times, then add primary antibody, incubate overnight at 4°C, then incubate in FITC-labeled goat anti-mouse secondary antibody at 37°C for 1.5h, then stain in propidium iodide at room temperature 10min, film production, Confocal microscope observation and photographing;
四、采用图像分析软件将同一发育时期的转基因羊和非转基因羊的卵母细胞进行荧光强度定量分析比较，选择每个发育时期都满足与非转基因羊卵母细胞相比荧光强度无显著性差异的转基因羊卵母细胞，即完成转基因羊卵母细胞的筛选。4. Use image analysis software to quantitatively analyze and compare the fluorescence intensity of oocytes of transgenic sheep and non-transgenic sheep at the same developmental stage, and select each developmental stage to meet the requirement that there is no significant difference in fluorescence intensity compared with non-transgenic sheep oocytes transgenic sheep oocytes, that is, the screening of transgenic sheep oocytes is completed.
步骤一中所述不同发育时期是指生发泡期(GerminalVesicle，GV期)和减数分裂Ⅱ期(MeiosisⅡ，MⅡ期)。The different development stages mentioned in the step 1 refer to the germinal vesicle (GV stage) and the meiosis II stage (Meiosis II, MII stage).
步骤三中一抗为5-甲基胞嘧啶。The primary antibody in step 3 is 5-methylcytosine.
步骤四所述图像分析软件为EZ-C1FreeViewer。The image analysis software described in step four is EZ-C1FreeViewer.
DNA甲基化(DNAMethylation)是真核生物的一种重要的最早发现的表观遗传修饰途径之一。DNA的甲基化修饰有多种方式，主要形成5-甲基胞嘧啶(5-mC)和少量的N6-甲基嘌呤(N6-mA)及7-甲基鸟嘌呤(7-mG)，其中最易发生和研究最为广泛的是发生在基因组CpG二核苷酸胞嘧啶上的甲基化，DNA甲基转移酶识别CpG二核苷酸，由S-腺苷甲硫氨酸(SAM)作为甲基供体，在基因组内CpG二核苷酸中胞嘧啶的5位碳原子加上一个甲基基团(5mC)。DNA methylation is one of the most important and earliest discovered epigenetic modification pathways in eukaryotes. There are many ways to modify DNA methylation, mainly forming 5-methylcytosine (5-mC) and a small amount of N6-methylpurine (N6-mA) and 7-methylguanine (7-mG), Among them, the most likely to occur and the most widely studied is the methylation of the genomic CpG dinucleotide cytosine, which is recognized by DNA methyltransferases and is formed by S-adenosylmethionine (SAM) As a methyl donor, a methyl group (5mC) is added to the carbon atom at position 5 of cytosine in a CpG dinucleotide in the genome.
哺乳动物一生中DNA甲基化水平经历2次显著变化，第一次发生在受精卵最初几次卵裂中，去甲基化酶清除了DNA分子上几乎所有从亲代遗传来的甲基化标志；第二次发生在胚胎植入子宫时，一种新的甲基化遍布整个基因组，甲基化酶使DNA重新建立一个新的甲基化模式。细胞内新的甲基化模式一旦建成，即可通过甲基化以“甲基化维持”的形式将新的DNA甲基化传递给所有子细胞DNA分子。The level of DNA methylation in mammals undergoes two significant changes in their lifetime. The first time occurs in the first few cleavages of fertilized eggs. Demethylases remove almost all the methylation marks inherited from the parents on the DNA molecules. the second time, when the embryo is implanted in the uterus, a new methylation spreads throughout the genome, and the methylase makes the DNA re-establish a new methylation pattern. Once the new methylation pattern in the cell is established, the new DNA methylation can be transmitted to all daughter cell DNA molecules through methylation in the form of "methylation maintenance".
由此可见，DNA甲基化水平对于生殖细胞的发育尤为重要，有文献报道，克隆和转基因家畜后代畸形率高，发育迟缓甚至死胎，是由于DNA的甲基化水平升高导致胚胎发育所必须的基因表达异常所造成的。It can be seen that the level of DNA methylation is particularly important for the development of germ cells. It has been reported in the literature that the offspring of cloned and transgenic livestock have a high rate of deformity, developmental delay and even stillbirth, which is necessary for embryonic development due to the increased level of DNA methylation. caused by abnormal gene expression.
现有的方法普遍采用显微镜来观察卵母细胞的形态来判断卵母细胞质量，这种方法受主观因素影响较大，而采用DNA的甲基化水平来评价卵母细胞的质量，从分子水平，特别是从表观遗传学的角度，来评价卵母细胞的质量，准确率更高，尤其是在克隆动物、转基因动物上的应用尤为重要，可有效避免产生死胎或者畸形后代。Existing methods generally use a microscope to observe the morphology of oocytes to judge the quality of oocytes. This method is greatly affected by subjective factors, and the methylation level of DNA is used to evaluate the quality of oocytes. From the molecular level , especially from the perspective of epigenetics, to evaluate the quality of oocytes, the accuracy rate is higher, especially in the application of cloned animals and transgenic animals, which can effectively avoid stillbirth or deformed offspring.
本发明方法中卵母细胞经免疫荧光染色后，用Confocal(荧光显微镜)采集图片，免疫荧光染色图片的荧光强度代表DNA的甲基化水平，软件EZ-C1FreeViewer可分析图片上的荧光强度(绿色荧光代表甲基化水平)，通过转基因和对照组(野生型羊)进行比较，通过统计分析软件进行分析，两组间荧光值强度(即DNA甲基化水平)无显著差异可认为卵母细胞具有正常的发育能力。In the method of the present invention, after the oocyte is stained by immunofluorescence, the picture is collected with Confocal (fluorescence microscope), the fluorescence intensity of the immunofluorescence staining picture represents the methylation level of DNA, and software EZ-C1FreeViewer can analyze the fluorescence intensity (green) on the picture Fluorescence represents the level of methylation), compared with the transgenic and control group (wild-type sheep), and analyzed by statistical analysis software, there is no significant difference in the intensity of fluorescence values (ie, DNA methylation level) between the two groups, which can be considered as oocyte have normal developmental capacity.
受到目前转基因技术水平的限制，外源基因的整合效率非常低，转基因家畜的数量非常稀少，所以转基因家畜的繁殖性能显的尤为重要，直接关系到转基因家畜自身健康状况和后代扩群效率。本发明方法的建立从分子水平直接评估卵母细胞的质量，为生产健康的转基因后代提供科学依据。所以该方法具有一定的科学性和实用性，是一种高效的转基因动物生物安全评价方法。Limited by the current level of transgenic technology, the integration efficiency of exogenous genes is very low, and the number of transgenic livestock is very rare, so the reproductive performance of transgenic livestock is particularly important, which is directly related to the health status of transgenic livestock and the efficiency of offspring expansion. The establishment of the method of the invention directly evaluates the quality of the oocyte from the molecular level, and provides a scientific basis for producing healthy transgenic offspring. Therefore, this method is scientific and practical, and it is an efficient biosafety evaluation method for transgenic animals.
附图说明Description of drawings
图1为实施例1中GV期卵母细胞甲基化免疫荧光染色图片；图2为实施例1中MⅡ期卵母细胞甲基化免疫荧光染色图片。FIG. 1 is a picture of immunofluorescence staining of methylation of oocytes at GV stage in Example 1; FIG. 2 is a picture of immunofluorescence staining of methylation of oocytes of stage MⅡ in Example 1.
具体实施方式Detailed ways
本发明技术方案不局限于以下所列举具体实施方式，还包括各具体实施方式间的任意组合。The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any combination of the specific embodiments.
具体实施方式一：本实施方式转基因羊卵母细胞的筛选方法，按以下步骤进行：Specific embodiment one: the screening method of the transgenic sheep oocyte of the present embodiment is carried out according to the following steps:
一、采用CIDR+FSH的方法对转基因羊和非转基因羊进行超数排卵，收集不同发育时期的卵母细胞；1. Using the method of CIDR+FSH to superovulate transgenic sheep and non-transgenic sheep, and collect oocytes at different developmental stages;
二、用体积百分浓度为4％的多聚甲醛固定卵母细胞至少30分钟，然后依次用体积百分浓度为1％的Triton-100处理30分钟、体积百分浓度为1％的HCl处理30分钟、2mg/mL的BSA封闭液封闭30分钟；2. Fix oocytes with 4% paraformaldehyde by volume for at least 30 minutes, and then treat them with 1% Triton-100 for 30 minutes and 1% HCl for 30 minutes. 30 minutes, 2mg/mL BSA blocking solution for 30 minutes;
三、将卵母细胞经PBS吹洗3次，然后加入一抗，4℃孵育过夜，然后于FITC标记的山羊抗小鼠二抗中37℃孵育1.5h，接着于碘化丙碇中室温染色10min，制片，Confocal显微镜观察并拍照；3. Wash oocytes with PBS 3 times, then add primary antibody, incubate overnight at 4°C, then incubate in FITC-labeled goat anti-mouse secondary antibody at 37°C for 1.5h, then stain in propidium iodide at room temperature 10min, film production, Confocal microscope observation and photographing;
四、采用图像分析软件将同一发育时期的转基因羊和非转基因羊的卵母细胞进行荧光强度定量分析比较，选择每个发育时期都满足与非转基因羊卵母细胞相比荧光强度无显著性差异的转基因羊卵母细胞，即完成转基因羊卵母细胞的筛选。4. Use image analysis software to quantitatively analyze and compare the fluorescence intensity of oocytes of transgenic sheep and non-transgenic sheep at the same developmental stage, and select each developmental stage to meet the requirement that there is no significant difference in fluorescence intensity compared with non-transgenic sheep oocytes transgenic sheep oocytes, that is, the screening of transgenic sheep oocytes is completed.
具体实施方式二：本实施方式与具体实施方式一不同的是：步骤一中所述不同发育时期是指生发泡期和减数分裂Ⅱ期。其它与具体实施方式一相同。Embodiment 2: This embodiment differs from Embodiment 1 in that: the different developmental stages mentioned in step 1 refer to the germinal vesicular stage and meiosis II stage. Others are the same as in the first embodiment.
卵母细胞根据发育阶段是分为GV、GVBD、MI、MII期，本实施方式中选取了GV和MII期卵母细胞，原因是1、这两个时期代表卵母细胞的典型发育时期，其甲基化水平正常完全说明卵母细胞质量正常；2、这两个时期的卵母细胞较容易获得，有利于实验的操作简易性。Oocytes are divided into GV, GVBD, MI, and MII stages according to the developmental stage. In this embodiment, the GV and MII stage oocytes are selected because 1. These two stages represent typical developmental stages of oocytes. A normal methylation level completely indicates that the quality of oocytes is normal; 2. Oocytes in these two stages are easier to obtain, which is conducive to the simplicity of the experiment.
具体实施方式三：本实施方式与具体实施方式一或二不同的是：步骤三中一抗为5-甲基胞嘧啶。其它与具体实施方式一或二相同。Embodiment 3: This embodiment differs from Embodiment 1 or Embodiment 2 in that: in Step 3, the primary antibody is 5-methylcytosine. Others are the same as in the first or second embodiment.
具体实施方式四：本实施方式与具体实施方式一至三之一不同的是：步骤四所述图像分析软件为EZ-C1FreeViewer。其它与具体实施方式一至三之一相同。Embodiment 4: This embodiment differs from Embodiment 1 to Embodiment 3 in that: the image analysis software described in Step 4 is EZ-C1FreeViewer. Others are the same as those in the first to third specific embodiments.
实施例1：Example 1:
本实施例转基因羊卵母细胞的筛选方法，按以下步骤进行：The screening method of the transgenic sheep oocyte of the present embodiment is carried out according to the following steps:
一、采用CIDR+FSH的方法对转TLR4基因羊和非转基因羊进行超数排卵，收集不同发育时期的卵母细胞；1. Using the method of CIDR+FSH to superovulate TLR4 transgenic sheep and non-transgenic sheep, and collect oocytes at different developmental stages;
二、用体积百分浓度为4％的多聚甲醛固定卵母细胞至少30分钟，然后依次用体积百分浓度为1％的Triton-100处理30分钟、体积百分浓度为1％的HCl处理30分钟、2mg/mL的BSA封闭液封闭30分钟；2. Fix oocytes with 4% paraformaldehyde by volume for at least 30 minutes, and then treat them with 1% Triton-100 for 30 minutes and 1% HCl for 30 minutes. 30 minutes, 2mg/mL BSA blocking solution for 30 minutes;
三、将卵母细胞经PBS吹洗3次，然后加入一抗，4℃孵育过夜，然后于FITC标记的山羊抗小鼠二抗中37℃孵育1.5h，接着于碘化丙碇中室温染色10min，制片，Confocal显微镜观察并拍照；3. Wash oocytes with PBS 3 times, then add primary antibody, incubate overnight at 4°C, then incubate in FITC-labeled goat anti-mouse secondary antibody at 37°C for 1.5h, then stain in propidium iodide at room temperature 10min, film production, Confocal microscope observation and photographing;
四、采用图像分析软件将同一发育时期的转TLR4基因羊和非转基因羊的卵母细胞进行荧光强度定量分析比较，选择每个发育时期都满足与非转基因羊卵母细胞相比荧光强度无显著性差异的转TLR4基因羊卵母细胞，即完成转基因羊卵母细胞的筛选。4. Use image analysis software to quantitatively analyze and compare the fluorescence intensity of oocytes of transgenic TLR4 sheep and non-transgenic sheep at the same developmental stage, and select each developmental stage to meet the fluorescence intensity compared with non-transgenic sheep oocytes. Transgenic sheep oocytes with sex difference TLR4 gene, that is, the screening of transgenic sheep oocytes is completed.
步骤一中所述不同发育时期是指生发泡期(GV期)和减数分裂Ⅱ期(MⅡ期)。The different development stages mentioned in step 1 refer to germinal vesicular stage (GV stage) and meiosis II stage (MII stage).
步骤三中一抗为5-甲基胞嘧啶。The primary antibody in step 3 is 5-methylcytosine.
步骤四所述图像分析软件为EZ-C1FreeViewer。The image analysis software described in step four is EZ-C1FreeViewer.
本实施例GV期卵母细胞甲基化免疫荧光染色图片如图1所示，MⅡ期卵母细胞甲基化免疫荧光染色图片如图2所示，图中TG表示转TLR4基因羊，WT表示非转基因羊，5-Mec为5甲基胞嘧啶，在图片显示绿色荧光，DNA就是细胞核，显示红色荧光，Merge是绿色荧光和红色荧光叠加的效果，其目的是为了表示DNA甲基化免疫荧光染色位点的正确性，即绿色荧光和红色荧光是完全重合的。In this example, the methylation immunofluorescent staining pictures of oocytes at the GV stage are shown in Figure 1, and the methylation immunofluorescent staining pictures of MⅡ stage oocytes are shown in Figure 2, in which TG represents TLR4 transgenic sheep, and WT represents Non-transgenic sheep, 5-Mec is 5-methylcytosine, green fluorescence is shown in the picture, DNA is the nucleus, showing red fluorescence, Merge is the effect of the superposition of green fluorescence and red fluorescence, and its purpose is to express DNA methylation immunofluorescence The correctness of the staining sites, that is, the green fluorescence and red fluorescence are completely coincident.
本实施例中转TLR4基因羊卵母细胞的甲基化水平和对照组(转基因羊)无显著差异(P>0.05)。表明筛选出的转TLR4基因羊具有正常的生殖和发育能力。There was no significant difference (P>0.05) in the methylation level of TLR4 gene-transferred sheep oocytes and the control group (transgenic sheep) in this example. It indicated that the selected TLR4 transgenic sheep had normal reproductive and developmental abilities.
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