CN102242151A - Method for producing transgenic rabbits with microscopic insemination technology - Google Patents
Method for producing transgenic rabbits with microscopic insemination technology Download PDFInfo
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- CN102242151A CN102242151A CN2011100935871A CN201110093587A CN102242151A CN 102242151 A CN102242151 A CN 102242151A CN 2011100935871 A CN2011100935871 A CN 2011100935871A CN 201110093587 A CN201110093587 A CN 201110093587A CN 102242151 A CN102242151 A CN 102242151A
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Abstract
The invention relates to a method for producing transgenic rabbits with a microscopic insemination technology. The method comprises the following steps: collecting a seminal fluid through a false vagina process or the seminal fluid from an epididymis, suspending with a PBS (phosphate buffer solution), packaging into a cryovial after centrifugation washing, and sending to liquid nitrogen for saving; taking the cryovial with sperms out of the liquid nitrogen, unfreezing at room temperature, adding an exogenous DNA plasmid seminal fluid for being fully mixed with the freeze-thaw sperms to make the final concentration of DNA plasmids to be 30 ng/mul, and incubating for 10 min at a temperature of 4 DEG C to allow the sperms to be fully combined with DNA before a microscopic operation; and then injecting the sperms which breed the DNA into rabbit oocyte cytoplasm through the microscopic insemination technology, and putting in a culture solution and culturing for 2-4 days after carrying out chemical activation processing on microscopic inseminated oocytes. Transgenic rabbit embryos are successfully produced through the method of the present invention, and the positive rate of the embryos reaches 59.65%. Female rabbits are pregnant through embryo transplant and bears live little rabbits, the little rabbits are subjected to cellular and molecular biology identification, and a case that four transgenic rabbits express the exogenous genes is proved, so the positive rate is 57.14%.
Description
Technical field
The invention belongs to Animal Biotechnology, particularly the directed genetic improvement of the meat of rabbit field.It specifically is a kind of method of utilizing the microscopic insemination technology to produce transgene rabbit.
Background technology
Rabbit is a kind of broad-spectrum economic animal and laboratory animal, and it is less to have a build, advantage such as reproduction speed is fast, with short production cycle.Yet the speed of growth of the present tame rabbit breeds of China is slow, dressing percentage is low, directly has influence on the economic benefit of rabbit keeping, is badly in need of using modern biotechnology it is improved, and cultivates the new species of rabbit that meat is good, meat productivity is high.Utilize the transgene clone technology to produce transgene rabbit,, after gene transfection and cultivating screening, carry out nuclear transplantation again and be difficult to obtain transgenic embryos because the somatic vigor of rabbit is low.And the microscopic insemination transgenic technology is exactly that foreign gene and sperm are combined by certain means external, make sperm carry marker gene or external source goal gene as a kind of carrier, by obtaining transgenic embryos, carry out obtaining the young rabbit of transgenosis after the embryo transfer again with Oocyte in Vitro microscopic insemination mode.This method has easy to operate advantage such as simple, and used sperm can be the sperm of living, and also can use the sperm after the freeze thawing.For this reason, the present invention passes through microscopic insemination technology produced in vitro transgene rabbit embryo, and sets up a kind of novel method of producing transgene rabbit in conjunction with embryo transfer technology.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of method of producing transgene rabbit by the microscopic insemination technology.
The present invention is by solving the problems of the technologies described above by the following technical solutions: a kind of method of utilizing the microscopic insemination technology to produce transgene rabbit comprises the steps:
(1) collection of seminal fluid and preservation: rabbit seminal fluid by the collection of artificial vagina method or the seminal fluid of collecting in the rabbit epididymis, with phosphoric acid buffer PBS upstream after half an hour, washed in centrifugal 5 minutes in 2500rpm, repeated washing three times is 1x10 with PBS liquid weaker concn again
6Individual sperm/milliliter divides to install to freeze pipe, and directly drop in the liquid nitrogen and preserve,
(2) hatching of sperm and foreign DNA: the freeze pipe that sperm will be housed takes out from liquid nitrogen, thaw under the room temperature, adding concentration is the foreign DNA plasmid of 100ng/ul and the abundant mixing of sperm of freeze thawing, the final concentration that makes the DNA plasmid is 30ng/ul, place under 4 ℃ of conditions and hatched 10 minutes, sperm is fully combined with DNA, obtain to have hatched the sperm of DNA
(3) preparation of microscopic insemination transgene rabbit: the sperm of having hatched DNA that will obtain is injected in the rabbit oocyte kytoplasm by the microscopic insemination technology, ovocyte behind the microscopic insemination is after ionomycin (Ion) and dimethylamino-purine (6-DMAP) associating cycloheximide (CHX) activates processing, put into to cultivate to drip and cultivated 2-4 days, the microscopic insemination embryo who obtains is detected the GFP expression under fluorescent microscope, filter out the microscopic insemination transgenic embryos that GFP expresses, and transplant and give acceptor female rabbit.
Outstanding advantage of the present invention is:
1, uses method of the present invention, successfully produced the transgene rabbit embryo, embryo's positive rate reaches 59.65% (312/523), through having 3 female rabbits to obtain gestation after the embryo transfer and giving birth to 7 of young rabbits alive, young rabbit is through the testing of a series of cell and molecular biology identification, the result shows 4 positive transgene rabbits, and positive rate is 57.14%.Illustrate and utilize this invention can successfully obtain the microscopic insemination transgene rabbit.
2, this method need not be added any frostproofer, can be placed on the medium-term and long-term preservation of liquid nitrogen, and room temperature is thawed available during use.
Description of drawings
Fig. 1 is the rabbit 4-cell stage transgenic embryos that expression that the present invention obtains has GFP.
Fig. 2 is rabbit transgenic embryos blastula stage that expression that the present invention obtains has GFP.
Fig. 3 is preceding rabbit 2, the 4-cell stage transgenic embryos that GFP is arranged of expressing of embryo transfer that the present invention obtains.
Fig. 4 is preceding rabbit transgenic embryos blastula stage that GFP is arranged of expressing of embryo transfer that the present invention obtains.
Fig. 5 is the photo of the transgene rabbit of 12 days December in 2010 birth that obtains of the present invention.
Fig. 6 is the photo of the transgene rabbit of being born on 01 06th, 2011 of 2 the present invention acquisition.
Fig. 7 is the western blotting detected result of the transgene rabbit that obtains of the present invention.
Fig. 8 is the southern blotting detected result of the transgene rabbit that obtains of the present invention.
Embodiment
By the following examples technical scheme of the present invention is described further.
A kind of method of utilizing the microscopic insemination technology to produce transgene rabbit comprises the steps:
1, the collection of seminal fluid and preservation: rabbit seminal fluid by the collection of artificial vagina method or the seminal fluid of collecting in the rabbit epididymis, with phosphoric acid buffer PBS upstream after half an hour, 2500rpm washed in centrifugal 5 minutes, and repeated washing three times is 1x10 with PBS liquid weaker concn
6Individual sperm/milliliter divides the freeze pipe that installs to 200ul, and every pipe 50ul directly drops in the liquid nitrogen and preserves.
2, the superovulation of female rabbit is handled: the single cage of the rabbit that experimental animal room is raised was raised 20 days, allow its free choice feeding, freely drink water, then it being carried out superovulation handles, adopt three days six times injections of measurement method FSH 1.2mg altogether, each 12 hours at interval, inject FSH for the last time and inject 100IU HCG after 12 hours, the HCG injection underwent surgery towards ovum after 14 hours, reclaimed the ovocyte in the uterine tube.
3, hatching of sperm and foreign DNA: the freeze pipe that sperm will be housed takes out from liquid nitrogen, thaw under the room temperature, adding concentration is the foreign DNA plasmid of 100ng/ul and the abundant mixing of sperm of freeze thawing, the final concentration of DNA plasmid is 30ng/ul, place under 4 ℃ of conditions and hatched 10 minutes, sperm is fully combined with DNA.
4, the preparation of microscopic insemination transgene rabbit: the rabbit oocyte that operation is recovered to is put into 0.15% Unidasa, the granulosa cell that is enclosed on the ovocyte zona pellucida is removed in piping and druming gently, the ovocyte that removes behind the granulosa cell is put into the operation liquid that concentration is 5ug/ml cytochalasin B (CB) with about 20 pieces every batch, in another adjacent drop, put into the sperm and PVP mixing liquid 10 microlitres of incubated dna, under inverted microscope (Japanese Nikon TCE300), sperm is injected into then and carries out the microscopic insemination operation in the ovocyte kytoplasm.Behind the EO, the immigration of the ovocyte behind the microscopic insemination is placed with in the culture plate of nutrient solution, puts into 38.5 ℃, 5%CO
2Incubator in cultivate, it is to cultivate 5 minutes in the 5 μ mol/L ionomycins (Ion) that cultivation moves into concentration with the ICSI embryo after half an hour, with nutrient solution washing three times, the actinomycetes that move into the dimethylamino-purine that concentration is 2mmol (6-DMAP) combined concentration again and be 5 μ g are with cultivating 1.5 hours in the nutrient solution of (CHX), activate processing, and then drip with moving into the cultivation that is placed with the 30ul embryo medium after the nutrient solution washing three times, at 38.5 ℃, 5%CO
2Incubator in cultivated 2-4 days, under fluorescent microscope, detect the GFP expression with cultivating the embryo who obtains, filter out transgenic embryos that GFP expresses and transplant female rabbit uterine tube or intrauterine to estrus synchronization, female rabbit obtains gestation, giving birth to young rabbit after wherein having the female rabbits of 3 acceptors through about one month the Gestation period has 7 and survives, young rabbit carries out transgenosis through PCR, frozen tissue section's laser scanning co-focusing detection, western blotting and southern blotting method and identifies that 4 positive transgene rabbits are arranged.
Embodiment 1
On November 9th, 2010, we produce 35 pieces of transgene rabbit embryos by the microscopic insemination technology, wherein there be 23 (65.7%) piece of 2 cell stage phase to express GFP, by operation positive embryo transfer is gone in the female rabbit uterine tube of acceptor, through after the gestation, give birth to 3 young rabbits on December 12nd, 2010 and all survive, detect 2 positive transgene rabbits through molecular biology method.
Embodiment 2
On December 7th, 2010, we are implanted into the positive embryo of 38 pieces of division to 2 cells respectively in female rabbit uterine tube of two estrus synchronizations, all obtain gestation, and in following 7 the young rabbits of symbiosis on January 6 in 2011, wherein survive 4, then, these 4 rabbits that survive are carried out the testing of a series of molecular biology identification, the result proves 2 positive transgene rabbits.
Claims (1)
1. a method of utilizing the microscopic insemination technology to produce transgene rabbit is characterized in that this method comprises the steps:
(1) collection of seminal fluid and preservation: rabbit seminal fluid by the collection of artificial vagina method or the seminal fluid of collecting in the rabbit epididymis, with phosphoric acid buffer PBS upstream after half an hour, washed in centrifugal 5 minutes in 2500rpm, repeated washing three times is 1x10 with PBS liquid weaker concn again
6Individual sperm/milliliter divides to install to freeze pipe, and directly drop in the liquid nitrogen and preserve,
(2) hatching of sperm and foreign DNA: the freeze pipe that sperm will be housed takes out from liquid nitrogen, thaw under the room temperature, adding concentration is the foreign DNA plasmid of 100ng/ul and the abundant mixing of sperm of freeze thawing, the final concentration that makes the DNA plasmid is 30ng/ul, place under 4 ℃ of conditions and hatched 10 minutes, sperm is fully combined with DNA, obtain to have hatched the sperm of DNA
(3) preparation of microscopic insemination transgene rabbit: the sperm of having hatched DNA that will obtain is injected in the rabbit oocyte kytoplasm by the microscopic insemination technology, ovocyte behind the microscopic insemination is after ionomycin (Ion) and dimethylamino-purine (6-DMAP) associating cycloheximide (CHX) activates processing, put into to cultivate to drip and cultivated 2-4 days, the microscopic insemination embryo who obtains is detected the GFP expression under fluorescent microscope, filter out the microscopic insemination transgenic embryos that GFP expresses, and transplant and give acceptor female rabbit.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107574183A (en) * | 2017-08-07 | 2018-01-12 | 苏州湖桥生物科技有限公司 | A kind of pronuclear microinjection method in transgenic rabbits mould manufacturing process |
| CN114606180A (en) * | 2022-04-06 | 2022-06-10 | 广东省第二人民医院（广东省卫生应急医院） | Method for improving pregnancy rate by acquiring high development potential sperms |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100293623A1 (en) * | 2002-01-10 | 2010-11-18 | Institut National De La Recherche Agronomique | Rabbit nuclear cloning method and uses thereof |
| CN101979585A (en) * | 2010-09-30 | 2011-02-23 | 广西大学 | Method for producing genetically modified rabbit |
-
2011
- 2011-04-14 CN CN2011100935871A patent/CN102242151A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100293623A1 (en) * | 2002-01-10 | 2010-11-18 | Institut National De La Recherche Agronomique | Rabbit nuclear cloning method and uses thereof |
| CN101979585A (en) * | 2010-09-30 | 2011-02-23 | 广西大学 | Method for producing genetically modified rabbit |
Non-Patent Citations (5)
| Title |
|---|
| 《Molecular Reproduction & Development》 20081205 Masumi Hirabayashi et al. Activation regimens for full-term development of rabbit oocytes injected with round spermatids 573-579 1 第76卷, 第6期 * |
| MASUMI HIRABAYASHI ET AL.: "Activation regimens for full‐term development of rabbit oocytes injected with round spermatids", 《MOLECULAR REPRODUCTION & DEVELOPMENT》 * |
| QY LI ET AL.: "Full-Term Development of Rabbit Embryos Produced by ICSI with Sperm Frozen in Liquid Nitrogen without Cryoprotectants", 《REPRODUCTION IN DOMESTIC ANIMALS》 * |
| 张延浩: "单精子注射法生产转基因家兔胚胎的研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 张颖 等: "异源Oct-4基因启动子在猪、兔和小鼠早期胚胎中的时空表达活性", 《生物化学与生物物理进展》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107574183A (en) * | 2017-08-07 | 2018-01-12 | 苏州湖桥生物科技有限公司 | A kind of pronuclear microinjection method in transgenic rabbits mould manufacturing process |
| CN114606180A (en) * | 2022-04-06 | 2022-06-10 | 广东省第二人民医院（广东省卫生应急医院） | Method for improving pregnancy rate by acquiring high development potential sperms |
| CN114606180B (en) * | 2022-04-06 | 2024-01-30 | 广东省第二人民医院（广东省卫生应急医院） | Method for improving pregnancy rate by acquiring sperm with high development potential |
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Application publication date: 20111116 |