CN104267011A - Transgenic sheep oocyte screening method - Google Patents
Transgenic sheep oocyte screening method Download PDFInfo
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- CN104267011A CN104267011A CN201410489943.5A CN201410489943A CN104267011A CN 104267011 A CN104267011 A CN 104267011A CN 201410489943 A CN201410489943 A CN 201410489943A CN 104267011 A CN104267011 A CN 104267011A
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Abstract
The invention relates to a transgenic sheep oocyte screening method, and relates to an oocyte screening method, which aims at solving the problems of the traditional transgenic sheep oocyte quality detection method that materials are difficult to get and the experimental period is long. The method comprises the following steps: I, performing superovulation on transgenic sheep and non-transgenic sheep, and collecting the oocyte in different development periods; II, fixing the oocyte by utilizing paraformaldehyde, and processing the oocyte sequentially by utilizing triton-100, HCl and BSA closed solutions; III, blowing the oocyte by virtue of PBS, adding a primary antibody, incubating the oocyte overnight, then incubating the oocyte in a secondary antibody, dying, carrying out film making, observing the oocyte by utilizing a microscope, and photographing the oocyte; and IV, performing fluorescence intensity quantitative analysis and comparison on the oocyte of the transgenic sheep and the non-transgenic sheep in the same development period, selecting the transgenic sheep oocyte which is not quite different from the non-transgenic sheep oocyte to complete the screening of the transgenic sheep oocyte. The method is used for screening the transgenic sheep oocyte.
Description
Technical field
The present invention relates to a kind of screening technique of egg mother cell.
Background technology
Since nineteen eighty-two, trangenic mice came out, transgenic animals achieve breakthrough progress in fields such as disease model, medicine, breed of variety, environmental protection and resource preservations.The continuous expansion of applying along with transgenic animals and people are to the worry of bio-safety and concern, and the research of transgenic animals bio-safety is arisen at the historic moment.The research of transgenic animals bio-safety based on the potential risk of transgenic animals and products thereof, it and genes of interest and mode of operation closely related.The genes of interest of transgenic animals and expression product, and in Long-Time Service and accumulative process, all likely bring new risk.For taking precautions against the risk of genetically modified organism to environment and human health, and promote the sound development of transgenosis industry, each link of Study on Transgenic Animal all through tight detecting and assessing, will take precautions against the factor that all are harmful to human health, animal health and Environmental security as much as possible.
DNA methylation is one of the most basic epigenetic modification mode, most important to mammiferous growth.DNA methylation refers under the effect of DNA methylation transferase (Dnmts), by S-adenosylmethionine (SAM) as methyl donor, in genome, in CpG dinucleotide, 5 carbon atoms of cytimidine add a methyl group (5mC).DNA methylation is divided into maintenance to methylate and from the beginning methyl two kinds of patterns, participates in much important growth event, such as, and gene expression regulation, genomic imprinting, x chromosome inactivation and heterochromatic formation.DNA methylation is most important for the Differentiation and development of Mammalian germ cells, genomic DNA methylation level is in dynamic reprogrammed process, main manifestations is that archaeocyte will experience a general demethylation process, and the genome of ripe gamete is then high methylation.After fertilization, also there is widely initiatively demethylation in zygotic gene group, after implanting, general newly methylating then occurs embryo.
By the transgenic sheep that micro-injection method is produced, the radom insertion of foreign gene, and genes of interest process LAN, the methylation level whether affecting egg mother cell it be unclear that.In addition, micromanipulation is also the key factor affecting egg mother cell methylation level.Because transgenic animals are rare, especially large livestock animals, therefore difficult from this angle estimator transgenic animals security of healthy reproduction, as difficulty of drawing materials, experimental period is long.We invent new appraisal procedure is egg mother cell by different development stage in super ovulation techniques acquisition volume, adopt the method for immunofluorescence dyeing, confocal is utilized to gather picture, detect F0 for transgenosis and non-transgenic sheep egg mother cell full-length genome methylation level difference, assess transgenic sheep Reproductive Health.
Summary of the invention
The present invention will solve in existing transgenic sheep Oocyte quality detection method to there is difficulty of drawing materials, and the problem that experimental period is long, provides a kind of screening technique of transgenic sheep egg mother cell.
The screening technique of transgenic sheep egg mother cell of the present invention, carries out according to the following steps:
One, adopt the method for CIDR+FSH to carry out superfecundation to transgenic sheep and non-transgenic sheep, collect the egg mother cell of different development stage;
Two, fix egg mother cell at least 30 minutes with the paraformaldehyde that concentration expressed in percentage by volume is 4%, then successively with Triton-100 process 30 minutes, concentration expressed in percentage by volume that concentration expressed in percentage by volume is 1% be the HCl process 30 minutes of 1%, the BSA confining liquid of 2mg/mL closes 30 minutes;
Three, by egg mother cell through PBS purge 3 times, then add primary antibodie, 4 DEG C of overnight incubation, then in the goat anti-mouse two of FITC mark is anti-37 DEG C hatch 1.5h, then room temperature dyeing 10min in propidium iodide, film-making, Confocal microscopic examination is also taken pictures;
Four, adopt image analysis software that the egg mother cell of the transgenic sheep of same developmental stage and non-transgenic sheep is carried out fluorescence intensity quantitative test to compare, select each developmental stage to meet the transgenic sheep egg mother cell of fluorescence intensity there was no significant difference compared with non-transgenic sheep egg mother cell, namely complete the screening of transgenic sheep egg mother cell.
Different development stage described in step one refers to germ-vesicle phase (GerminalVesicle, GV phase) and meiosis II phase (Meiosis II, M II phase).
In step 3, primary antibodie is 5-methylcytosine.
Image analysis software described in step 4 is EZ-C1FreeViewer.
DNA methylation (DNAMethylation) is one of Eukaryotic a kind of important epigenetic modification approach found the earliest.Methylating of DNA is modified with various ways, main formation 5-methylcytosine (5-mC) and a small amount of N6-methyl purine (N6-mA) and 7-methyl guanine (7-mG), wherein the most easily occurring and studying is occur in methylating on genome CpG dinucleotide cytimidine the most widely, dnmt rna identification CpG dinucleotide, by S-adenosylmethionine (SAM) as methyl donor, in genome, in CpG dinucleotide, 5 carbon atoms of cytimidine add a methyl group (5mC).
Mammal in life DNA methylation level experiences 2 marked changes, and first time occurs in the initial spilting of an egg several times of embryonated egg, and demethylase removes nearly all mark that methylates come from parental generation heredity on DNA molecular; Second time is when occurring in Embryonic limb bud cell uterus, a kind of methylating and spread all over whole genome newly, and methylase makes DNA re-establish a new methylation patterns.New DNA methylation, once build up, is namely passed to all daughter cell DNA moleculars by methylating with the form of " methylate maintenance " by methylation patterns new in cell.
As can be seen here, DNA methylation level is particularly important for the growth of reproduction cell, has bibliographical information, clone and breeding transgenic livestock offspring abnormal rate high, hypoevolutism is stillborn foetus even, is because the methylation level rising of DNA causes the necessary abnormal gene expression of embryonic development to cause.
Existing method generally adopts microscope to observe the form of egg mother cell to judge Oocyte quality, this method affects larger by subjective factor, and adopt the methylation level of DNA to evaluate the quality of egg mother cell, from molecular level, particularly from the angle of epigenetics, evaluate the quality of egg mother cell, accuracy rate is higher, especially the application on cloned animal, transgenic animals is particularly important, can effectively avoid producing stillborn foetus or lopsided offspring.
In the inventive method, egg mother cell is after immunofluorescence dyeing, picture is gathered with Confocal (fluorescent microscope), the methylation level of the fluorescence intensity representation DNA of immunofluorescence dyeing picture, software EZ-C1FreeViewer can analyze the fluorescence intensity (green fluorescence represents methylation level) on picture, compared by transgenosis and control group (wild type sheep), analyzed by statistical analysis software, between two groups, without significant difference, fluorescent value intensity (i.e. DNA methylation level) can think that egg mother cell has normal developmental potency.
Be subject to the restriction of transgenic technology level up till now, the integration efficiency of foreign gene is very low, the quantity of breeding transgenic livestock is very rare, so aobvious particularly important of the reproductive performance of breeding transgenic livestock, is directly connected to breeding transgenic livestock self health status and offspring's group expanding efficiency.The quality of egg mother cell is directly assessed in the foundation of the inventive method from molecular level, provide scientific basis for producing healthy transgenic progeny.Having certain science and practicality in this way, is a kind of transgenic animals bio-safety evaluation method efficiently.
Accompanying drawing explanation
Fig. 1 is that in embodiment 1, GV phase egg mother cell methylates immunofluorescence dyeing picture; Fig. 2 is that in embodiment 1, M II phase egg mother cell methylates immunofluorescence dyeing picture.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the combination in any between each embodiment.
Embodiment one: the screening technique of present embodiment transgenic sheep egg mother cell, carry out according to the following steps:
One, adopt the method for CIDR+FSH to carry out superfecundation to transgenic sheep and non-transgenic sheep, collect the egg mother cell of different development stage;
Two, fix egg mother cell at least 30 minutes with the paraformaldehyde that concentration expressed in percentage by volume is 4%, then successively with Triton-100 process 30 minutes, concentration expressed in percentage by volume that concentration expressed in percentage by volume is 1% be the HCl process 30 minutes of 1%, the BSA confining liquid of 2mg/mL closes 30 minutes;
Three, by egg mother cell through PBS purge 3 times, then add primary antibodie, 4 DEG C of overnight incubation, then in the goat anti-mouse two of FITC mark is anti-37 DEG C hatch 1.5h, then room temperature dyeing 10min in propidium iodide, film-making, Confocal microscopic examination is also taken pictures;
Four, adopt image analysis software that the egg mother cell of the transgenic sheep of same developmental stage and non-transgenic sheep is carried out fluorescence intensity quantitative test to compare, select each developmental stage to meet the transgenic sheep egg mother cell of fluorescence intensity there was no significant difference compared with non-transgenic sheep egg mother cell, namely complete the screening of transgenic sheep egg mother cell.
Embodiment two: present embodiment and embodiment one unlike: different development stage described in step one refers to germ-vesicle phase and meiosis II phase.Other is identical with embodiment one.
Egg mother cell is be divided into GV, GVBD, MI, MII phase according to the stage of development, GV and MII phase egg mother cell is have chosen in present embodiment, the typical developmental stage that reason is 1, these two represent egg mother cell period, its methylation level normally illustrates that Oocyte quality is normal completely; 2, the egg mother cell in these two periods is easier to obtain, and is conducive to the ease of handling of testing.
Embodiment three: present embodiment and embodiment one or two unlike: in step 3, primary antibodie is 5-methylcytosine.Other is identical with embodiment one or two.
Embodiment four: one of present embodiment and embodiment one to three unlike: image analysis software described in step 4 is EZ-C1FreeViewer.Other is identical with one of embodiment one to three.
Embodiment 1:
The screening technique of the present embodiment transgenic sheep egg mother cell, carries out according to the following steps:
One, adopting the method for CIDR+FSH to turning TLR4 gene sheep and non-transgenic sheep carries out superfecundation, collecting the egg mother cell of different development stage;
Two, fix egg mother cell at least 30 minutes with the paraformaldehyde that concentration expressed in percentage by volume is 4%, then successively with Triton-100 process 30 minutes, concentration expressed in percentage by volume that concentration expressed in percentage by volume is 1% be the HCl process 30 minutes of 1%, the BSA confining liquid of 2mg/mL closes 30 minutes;
Three, by egg mother cell through PBS purge 3 times, then add primary antibodie, 4 DEG C of overnight incubation, then in the goat anti-mouse two of FITC mark is anti-37 DEG C hatch 1.5h, then room temperature dyeing 10min in propidium iodide, film-making, Confocal microscopic examination is also taken pictures;
Four, adopt image analysis software that the egg mother cell turning TLR4 gene sheep and non-transgenic sheep of same developmental stage is carried out fluorescence intensity quantitative test to compare, select each developmental stage meet compared with non-transgenic sheep egg mother cell fluorescence intensity there was no significant difference turn TLR4 gene sheep egg mother cell, namely complete the screening of transgenic sheep egg mother cell.
Different development stage described in step one refers to germ-vesicle phase (GV phase) and meiosis II phase (M II phase).
In step 3, primary antibodie is 5-methylcytosine.
Image analysis software described in step 4 is EZ-C1FreeViewer.
The present embodiment GV phase egg mother cell methylates immunofluorescence dyeing picture as shown in Figure 1, M II phase egg mother cell methylates immunofluorescence dyeing picture as shown in Figure 2, in figure, TG represents and turns TLR4 gene sheep, WT represents non-transgenic sheep, 5-Mec is 5 methylcysteins, at picture display green fluorescence, DNA is exactly nucleus, display red fluorescence, Merge is the effect of green fluorescence and red fluorescence superposition, its objective is the correctness in order to represent DNA methylation immunofluorescence dyeing site, namely green fluorescence and red fluorescence overlap completely.
The methylation level of the present embodiment transfer TLR4 gene sheep egg mother cell and control group (transgenic sheep) are without significant difference (P>0.05).Show that the TLR4 gene sheep that turns filtered out has normal reproduction and development ability.
Claims (4)
1. a screening technique for transgenic sheep egg mother cell, is characterized in that the method is carried out according to the following steps:
One, adopt the method for CIDR+FSH to carry out superfecundation to transgenic sheep and non-transgenic sheep, collect the egg mother cell of different development stage;
Two, fix egg mother cell at least 30 minutes with the paraformaldehyde that concentration expressed in percentage by volume is 4%, then successively with Triton-100 process 30 minutes, concentration expressed in percentage by volume that concentration expressed in percentage by volume is 1% be the HCl process 30 minutes of 1%, the BSA confining liquid of 2mg/mL closes 30 minutes;
Three, by egg mother cell through PBS purge 3 times, then add primary antibodie, 4 DEG C of overnight incubation, then in the goat anti-mouse two of FITC mark is anti-37 DEG C hatch 1.5h, then room temperature dyeing 10min in propidium iodide, film-making, Confocal microscopic examination is also taken pictures;
Four, adopt image analysis software that the egg mother cell of the transgenic sheep of same developmental stage and non-transgenic sheep is carried out fluorescence intensity quantitative test to compare, select each developmental stage to meet the transgenic sheep egg mother cell of fluorescence intensity there was no significant difference compared with non-transgenic sheep egg mother cell, namely complete the screening of transgenic sheep egg mother cell.
2. the screening technique of a kind of transgenic sheep egg mother cell according to claim 1, is characterized in that different development stage described in step one refers to germ-vesicle phase and meiosis II phase.
3. the screening technique of a kind of transgenic sheep egg mother cell according to claim 1, is characterized in that in step 3, primary antibodie is 5-methylcytosine.
4. the screening technique of a kind of transgenic sheep egg mother cell according to claim 1, is characterized in that image analysis software described in step 4 is EZ-C1FreeViewer.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110246135A (en) * | 2019-07-22 | 2019-09-17 | 新名医(北京)科技有限公司 | Monitor Follicles method, apparatus, system and storage medium |
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| CN102199595A (en) * | 2010-03-24 | 2011-09-28 | 香港城市大学 | Expression box, transgenic fish and uses thereof |
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Patent Citations (5)
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| CN1726393A (en) * | 2002-12-18 | 2006-01-25 | 约翰内斯·科伊 | Compounds and methods for detection of carcinomas and their precursor lesions |
| US20110088102A1 (en) * | 2008-05-28 | 2011-04-14 | Mitchell Turker | Methods of identifying inhibitors of gene silencing in mammalian cells |
| US20100081582A1 (en) * | 2008-10-01 | 2010-04-01 | Pioneer Hi-Bred International, Inc. | High throughput method for measuring total fermentables |
| CN102325791A (en) * | 2009-01-12 | 2012-01-18 | 乌拉·博纳斯 | Moudle type DNA binding domains and method of use |
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| CN110246135A (en) * | 2019-07-22 | 2019-09-17 | 新名医(北京)科技有限公司 | Monitor Follicles method, apparatus, system and storage medium |
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Application publication date: 20150107 |