CN102533827A - Method for preparing transgenic pig - Google Patents
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- CN102533827A CN102533827A CN2011103855629A CN201110385562A CN102533827A CN 102533827 A CN102533827 A CN 102533827A CN 2011103855629 A CN2011103855629 A CN 2011103855629A CN 201110385562 A CN201110385562 A CN 201110385562A CN 102533827 A CN102533827 A CN 102533827A
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Abstract
The invention discloses a method for preparing a transgenic pig, which is characterized by comprising the following steps: (a) preparing a carrier with foreign genes and a liposome solution into a mixed solution; (b) injecting the mixed solution into testis of an adult pig, wherein injection points are located at the positions of two sides of about 1.4 to 1.6cm away from a central line of the testis, and the injection length is about 1.4 to 1.6cm; and (c) enabling a boar with the injected testis to mate with a rutting sow after two months, wherein a generated piglet is the transgenic pig. Compared with random injection, by means of the method, the specific injection points and the injection depth are selected so as to be capable of greatly improving transgenic efficiency.
Description
Technical field:
The present invention relates to a kind of method for preparing transgenic pig, particularly liposome-mediated preparation transgenic pig method.
Background technology:
Transgenic technology starts from the eighties in 20th century, and at present, transgenic technology mainly contains micro-procaryotic injection method; The retroviral infection method, embryonic stem cell mediated method, somatic cell nuclear transfer technique; Intracytoplasmic sperm injection method in the sperm vector method, endochylema, ovocyte support methods etc.
The sperm vector method is with the carrier of sperm as foreign gene; In fertilization process, foreign gene is imported animal embryo; And foreign DNA is incorporated in the karyomit(e); Thereby foreign gene is got in the genome of filial generation at present; Using sperm is that carrier mediated transgenosis acquisition transgenic animal are mainly realized through following approach: the interior sperm infection protocol external sperm infection protocol of external sperm infection protocol and body can be divided into sperm and foreign DNA again and directly hatch method altogether, external electroporation introductory technique and liposome-mediated infection protocol; The sperm infection protocol can be divided into vas deferens injection infection protocol, convoluted seminiferous tubule injection infection protocol, testis injection infection protocol etc. again in the body.1971, Bracket etc. confirmed that first sperm has absorption and combines the foreign DNA ability, and foreign DNA is carried the entering ovocyte at the time of fertilization.1992; This method of application such as Lavitrano obtains positive transgenic mice; Concurrent ready-made ripe sperm head has the ability that absorbs foreign DNA; Its absorption region is to have specificly, and this zone is positioned at the acrosome back zone (post-acrosomal region) of sperm head, promptly near the zone of sperm nucleus.Shen Xinming etc. the directly enhanced green fluorescence protein expression vector of personnel selection cytomegalovirus promoter regulation and control are expelled in the convoluted seminiferous tubule of mouse, at last through with female mouse mating, successfully obtained the transgenic newborn mouse of expressing green fluorescent protein.Reports such as Yuan Jin are to through injecting foreign DNA in convoluted seminiferous tubule; And then combine convoluted seminiferous tubule electric shock or electroporation are handled; So that foreign gene gets into testis spermatogenic cell then with male mouse and female mouse mating of these processing, there is one group positive detection rate to reach 25% in the mouse that obtains.Shen etc. then more simplify treatment process, and at first directly the foreign DNA of green fluorescence expression and the medium of the inferior maple of dimethyl-are carried in injection in male rabbit testis, make DNA can get into testicular cell and androgone.With male rabbit and the doe mating handled, the offspring's that produces 56% young rabbit can be expressed the foreign gene that is imported efficiently after one month.This positive rate that changes foreign gene over to is much higher than the result of sperm vector method in the past or other non-fixed point transgenic methods.Li Bichun etc. are mark with the green fluorescence protein gene of reorganization, and upward (spermatogonial stem cells SSCs) has successfully produced transgenic chicken with the method that in-vitro transfection SSCs transplants again to the chicken of being in first through adopting transfection of spermatogonial stem cell in the cock testis; And be in duck and tame chicken go up the transfection functional gene and verify that the result explains that all this method need not special instrument requirement, can accomplish the process of transfection foreign gene; Method is simple, and is easy to operate, and efficient is high; Cost is low; Can produce the transgenic chicken crowd in large quantity, being based upon on the poultry of this method is significant, has broken through the bottleneck that restriction poultry transgenic is produced; Solved the difficult problem of poultry transgenic; The particularly in-vitro transfection success of transplanting has not only solved the difficult problem of poultry transgenic, and has opened up more wide application prospect for the application of adult stem cell.
Pre-treatment before sperm is hatched in the sperm vector method makes the fertility of sperm descend usually; So the auxiliary procreation technology ICSI that many scholars attempt using for reference medically makes the fertilization of processing sperm; Micro-fertilization technology is by the micrurgy appearance sperm or androgone directly to be injected in the ovocyte matter in the sperm kytoplasm, thereby accomplishes the process of fertilization.It has reduced the requirement to the various indexs of sperm, makes the various ovum fertilizations that normal fertilization can not take place in vivo and in vitro, can avoid polyspermy simultaneously, also valid approach is provided 1999 years for fertilization between the research xenogenesis.[44] people such as U.S. scientist Perry are applied to animal transgenic field with the ICSI technology first; Successfully obtained transgenic mice immediately; Investigators also carry out the genetically modified trial of ICSI respectively on the macaque pig; Embryo's transgenic positive rate is also very high, however transgenic animal that these experiments finally do not obtain being born 2006, and Kurom etc. are hatched sperm and the dna sequence dna segment that carries human albumin (hALB) gene and green fluorescent protein (GFP) gene altogether; Producing transgenic piggy Garc ' a-Va ' zquez that contains hALB and GFP gene etc. after the embryo transfer of producing through the ICSI method handles the sperm of pig through different methods; (frozen-thawing FT), does not add the cryoprotectant quick-frozen and handles (quick freezing without cryoprotectant agents through freeze thawing treatment in discovery; QF) and with the sperm that Triton X-100 (TX-100) handled compare with fresh sperm; Their combine the ability of foreign DNA obviously to strengthen, handle sperm with TX-100 and cause the damage of spermatid film more can promote sperm to combine to test also discovery with foreign DNA ground but its developmental potency reduces QF with the damage that FT causes, with fresh sperm and with the sperm of FT and TX-100 processing in the embryo of ICSI growth; Embryo's positive rate that EGFP expresses is close (to be respectively 37.04 ± 3.52%; 43.54 ± 5.41% and 29.03 ± 8.29%), and the sperm of handling through QF, EGFP embryo's positive rate obviously improves (80.43 ± 5.91%).This cytolemma that shows sperm plays crucial effects in DNA interacts; The spermatid film of treated mistake more helps foreign DNA and combines with the staining of sperm body; But handle through QF and TX-100, the nucleus of possible major injury sperm causes that DNA is broken; Or it is cause chromosomal breakage, thereby harmful to embryo's growth in the future.
At present, transgenic technology mainly contains DNA microinjection, embryonic stem cell mediated method, retroviral vector method, sperm vector method etc.Can effectively foreign gene be imported in the target cell with these methods, and obtain corresponding transgenic animal.The successful utilization of these technology has disclosed and produced the animal that carries foreign DNA is possible, but also there is some limitation in these gene transfer methods.Complicated like: microinjection schedule of operation, big to embryo's damage, high to technology, equipment requirements, particularly the poor efficiency (being lower than 1%) on the large mammal of development and application values is arranged cattle and sheep pig etc., greatly restricted this The Application of Technology prospect; Though the efficient of retroviral vector method transgenosis is higher; It is at random that but gene inserts; And the finite capacity of retroviral vector only can carry the exogenous dna fragment less than 10Kb, and retrovirus mainly infect body early embryo enliven the separation period cell; Thereby obtain be mostly chimeric animal, screen, cultivate, build frenulum for the downstream of transgenic animal and come very big human and material resources, financial resources to bear; In addition, retrovirus is prone to the pathogen gene sequence is introduced host genome, and the biological safety of animal receives all to arrive query.And sperm vector method less stable, repeatability is lower.Common testis injection method is owing to the direct injection vector plasmid, and transgene efficiency is lower, and unstable result.In sum, existing transgenic technology all exists cost high, and investment is big, and technical requirements is high, complicated operation, shortcoming such as transgene efficiency is lower.So, seek a kind of saving time, transgenic method laborsaving and that efficient is higher is extremely urgent.
Summary of the invention:
Of the present inventionly on sperm vector method basis, set up, through to spermatogonium transfection foreign gene, and then obtain to carry the sperm of goal gene; This friendship or artificial insemination then; Obtain transgenic pig,, can improve genetically modified efficient greatly according to technical scheme of the present invention.In order to realize the object of the invention, intend and adopt following technical scheme:
One aspect of the present invention relates to a kind of method for preparing transgenic pig, it is characterized in that comprising the steps: that carrier and liposome solutions that (a) will carry foreign gene process mixing solutions; (b) mixing solutions is expelled in the testis of the pig that grows up, wherein injection point is positioned at the position about testis medullary ray both sides 1.4-1.6cm, and injection depth is about 1.4-1.6cm; (c) after two months the boar of testis injection carry out this friendship with the sow that oestruses, the pig that farrows is transgenic pig.
In a preferred implementation of the present invention, described injection point is positioned at the position about testis medullary ray both sides 1.5cm, and injection depth is about 1.5cm.
In another one preferred implementation of the present invention, comprise that also the farrowing pig is carried out expression conditions to be detected with the screening transgenic pig.
In a preferred implementation of the present invention, respectively comprise at least 3 injection site in testis medullary ray both sides, be preferably 5 injection site.
In a preferred implementation of the present invention, above-mentioned preparation transgenic pig method is non-therapeutic purpose and/or non-diagnostic purpose.
Through method of the present invention,, select specific injection site and injection depth can greatly improve genetically modified efficient than injection at random.
Embodiment:
Material: liposome; The tincture of iodine; Stabilize injection, carry the vector plasmid pEGFP-N1-pGH carrier of foreign gene, reporter gene is EGFP gene-enhanced green fluorescence protein gene, and goal gene is pGH gene-pig growth hormone gene.
Step:
1. with 6ml concentration be: 400-500ng/ μ l left and right sides plasmid vector and 6ml liposome (LipofectamineTM2000) solution mix, and hatch half hour under the room temperature;
2. the boar that will grow up before the testis injection anaesthetizes or binds, treat its stable after, with cotton ball soaked in alcohol with pig testis surface skin wiped clean;
3. the position about testis medullary ray both sides 1.5cm is respectively selected 5 injection points at random; Draw 600 microlitre carriers and the liposome mixture is expelled in the pig testis tissue with the 1ml syringe; The syringe needle depth of penetration is confirmed according to pig build size, approximately about 1.5cm, is pulled outwardly pin while inject; After injection finishes syringe needle is rested in the testis tissue about 30s, prevent the liquid outflow;
4. after two months the boar of testis injection carry out this friendship with the sow that oestruses; Wait to farrow after detect its expression of gene situation; Through detecting, 43 positive transgenic pigs of piglet are arranged in 100 piglets, the ratio that the piglet that promptly has a foreign gene accounts for all piglets is about 43%.
When being understood that, above-mentioned embodiment only is an illustrative, concerning those of ordinary skills, can improve or conversion according to above-mentioned explanation, and all these improvement and conversion all should belong to the protection domain of accompanying claims of the present invention.
Claims (5)
1. method for preparing transgenic pig is characterized in that comprising the steps: that carrier and liposome solutions that (a) will carry foreign gene process mixing solutions; (b) mixing solutions is expelled in the testis of the pig that grows up, wherein injection point is positioned at the position about testis medullary ray both sides 1.4-1.6cm, and injection depth is about 1.4-1.6cm; (c) after two months the boar of testis injection carry out this friendship with the sow that oestruses, the pig that farrows is transgenic pig.
2. preparation transgenic pig method according to claim 1, described injection point is positioned at the position about testis medullary ray both sides 1.5cm, and injection depth is about 1.5cm.
3. preparation transgenic pig method according to claim 1 comprises that also the farrowing pig is carried out expression conditions to be detected with the screening transgenic pig.
4. preparation transgenic pig method according to claim 1 respectively comprises at least 3 injection site in testis medullary ray both sides, is preferably 5 injection site.
5. according to any described preparation transgenic pig method of claim 1-4, said preparation transgenic pig method is non-therapeutic purpose and/or non-diagnostic purpose.
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| CN201110385562.9A CN102533827B (en) | 2011-11-29 | 2011-11-29 | A kind of method preparing transgenic pig |
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| CN201110385562.9A CN102533827B (en) | 2011-11-29 | 2011-11-29 | A kind of method preparing transgenic pig |
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| CN102533827A true CN102533827A (en) | 2012-07-04 |
| CN102533827B CN102533827B (en) | 2016-02-17 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103352072A (en) * | 2013-05-07 | 2013-10-16 | 东北农业大学 | Transgene stock boar selection method and applications thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102181462A (en) * | 2011-02-24 | 2011-09-14 | 中国农业科学院北京畜牧兽医研究所 | New method for improving transgenic efficiency of animals |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102181462A (en) * | 2011-02-24 | 2011-09-14 | 中国农业科学院北京畜牧兽医研究所 | New method for improving transgenic efficiency of animals |
Non-Patent Citations (3)
| Title |
|---|
| 刘羞菲等: "《脂质体包裹质粒直接打点注射睾丸和卵巢生产转基因小鼠的研》", 《四川农业大学硕士学位论文》 * |
| 吴明明等: "《睾丸注射转染外源DNA制备转基因猪的研究》", 《第十六次全国动物遗传育种学术讨论会暨纪念吴仲贤先生诞辰100周年大会》 * |
| 肖红卫等: "精子介导生产转hCD59基因猪", 《华中农业大学学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103352072A (en) * | 2013-05-07 | 2013-10-16 | 东北农业大学 | Transgene stock boar selection method and applications thereof |
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Inventor after: Sun Jinhai Inventor after: Wu Mingming Inventor after: Cao Yuesheng Inventor after: Sun Jinning Inventor after: Liu Huanqi Inventor before: Sun Renyuan Inventor before: Zhang Yuanfa Inventor before: Lin Li Inventor before: Hu Aimei Inventor before: Chen Dong |
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Free format text: CORRECT: INVENTOR; FROM: SUN RENYUAN ZHANG YUANFA LIN LI HU AIMEI CHEN DONG TO: SUN JINHAI WU MINGMING CAO YUESHENG SUN JINNING LIU HUANQI |
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