CN102174577A - Method for producing genetically modified water buffalos by utilizing lentiviral vector - Google Patents
Method for producing genetically modified water buffalos by utilizing lentiviral vector Download PDFInfo
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- CN102174577A CN102174577A CN2011100253374A CN201110025337A CN102174577A CN 102174577 A CN102174577 A CN 102174577A CN 2011100253374 A CN2011100253374 A CN 2011100253374A CN 201110025337 A CN201110025337 A CN 201110025337A CN 102174577 A CN102174577 A CN 102174577A
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Abstract
The invention relates to a method for producing genetically modified water buffalos by utilizing a lentiviral vector, comprising the following steps: carrying out package and concentration on high-titer lentiviral, infecting and screening fetal fibroblast of a water buffalo; using a microinjection method to prepare a genetically modified DNAchimeric embryo of the water buffalo; transplating a positive embryo to the receptor water buffalo. By utilizing the method provided by the invention, exogenous-gene water buffalo fibroblast, DNAchimeric blastaea of the genetically modified water buffalo, more than 30% of somatic cell expression exogenous genes and more than 40% of blastaea expression exogenous genes are obtained and a genetically modified water buffalo with green-fluorescent-protein modified genes is obtained.
Description
Technical field
The present invention relates to Animal Biotechnology, specifically be a kind of method of utilizing lentiviral vectors production transgenosis buffalo.
Background technology
Buffalo is one of main big domestic animal in China south, and biological characteristics such as have that adaptability is strong, high-temp resisting high-humidity resisting, disease resistance are strong, crude feed tolerance, working life are long is fit to rural area, China south and raises very much.Development and use buffalo poultry kind of resource is converted into breast with existing labour with buffalo and uses or newborn dualpurpose commodity buffalo, is the trend of buffalo herding industry development.Yet at present the breeding potential production performance very low, population of buffalo is inferior, is badly in need of development and improves a collection ofly improving buffalo reproductivity and milk yield with the closely-related agriculture hi-tech of domestic animal genetic improvement.Utilize the embryo engineering technology, particularly the transgenic animal technology is an effective technology measure of cultivating the animal new variety and cultivating the animal disease resistant new variety.2002, people were applied to Study on Transgenic Animal with the lentiviral vectors method first, and have obtained success multiple animals such as pig, oxen.The lentiviral vectors transgenosis has the following advantages: can infect somatoblast and Unseparated Cell; The multiple tissue of energy transfection comprises the body early embryo cell; Energy stable integration and long-term expression external source goal gene; Be difficult for bringing out host immune response; Can compatible a plurality of promotors etc.These advantages make the lentiviral vectors method become a new breakthrough on the transgenic animal methodology.With respect to other species, buffalo somatic cell and embryo are difficult for being infected by foreign gene, and the efficient of gene transfection is lower, and simultaneously, the external source goal gene is difficult for stable integration and long-term expression; This has increased difficulty for obtaining buffalo transgenic embryos and production transgenosis buffalo.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of method of utilizing lentiviral vectors production transgenosis buffalo, to adapt to the needs of buffalo breed improvement.
The present invention solves the problems of the technologies described above with following technical scheme: a kind of method of utilizing lentiviral vectors production transgenosis buffalo comprises the steps:
1, high titre slow virus packing: 293T cell that adopting goes down to posterity cultivates and 3 plasmid packaging systems and calcium phosphate transfection method are carried out the slow virus packing.Before the transfection, with the 293T cell with 1 * 10
5The density of cells/mL goes down to posterity in the culture dish of handling through poly-lysine, is 5%CO with the 10%DMEM nutrient solution in volume percent
237 ℃ of incubators in cultivate, standby when growing into 70～80% degree of converging.1h before the transfection is replaced with the unparalleled anti-DMEM of serum-free with substratum.
During transfection, be that 15 μ g vector plasmids, 10 μ g envelope protein plasmid p Δ 8.9,5 μ g packaging plasmid pVSV-G and 125 μ LCaCL2 mix in test tube, supply volume to 500 μ L 3 plasmids.With 500 μ L phosphoric acid disodium hydrogens, pH value is that 6.95 conversion fluid is added dropwise in the above-mentioned mixed solution, and room temperature leaves standstill 30min.The plasmid mixed solution dropwise being added in the 293T cell of 100mm culture dish, is 5%CO in volume percent
237 ℃ of incubators in cultivate, changed normal nutrient solution in second day and cultivate.48～72h collects virus after the transfection.In the centrifugal 10min of 1500rpm, 0.45 μ m filter filters collects virus stock solution used with the supernatant collected.Viral supernatant with 20000rpm centrifugal force, 4 ℃ of centrifugal 2h, is obtained concentrating slow virus ,-80 ℃ of freezing preservations with the PBS of 100 μ L is resuspended.
The detection of virus titer: the 293T cell is with concentration 2 * 10
5Cells/mL is inoculated in 24 orifice plates, and every hole adds 500 μ L DMEM nutrient solution overnight incubation.During detection 4 μ L virus liquid is added to 500 μ L and contains in the DMEM nutrient solution of 2%FBS and mix, change original nutrient solution.48～72h observes egfp expression situation and counting.
2, make buffalo transgenosis somatocyte: the buffalo inoblast of cultivating for 4～5 generations of going down to posterity infects with slow virus stoste.During cells infected,, add 0～10 μ g/ml polybrene except that with DMEM and 10% foetal calf serum.Transfection the 2nd day is changed normal nutrient solution and is continued culturing cell and go down to posterity detection green fluorescent protein GFP expression under fluorescent microscope 3 times.
3, make buffalo transgenosis chimeric embryo: adopt micro-injection method will concentrate slow virus and be injected under the buffalo mature oocyte zona pellucida or in the IVF zygote.
Buffalo Oocyte in Vitro after fertilization, slow virus concentrated solution 30～70pL is expelled to buffalo IVF zygote, after cultivating 7～9 days altogether outward with the individual layers of granulosa cell, detect blastaea GFP expression under fluorescent microscope, screening obtains buffalo transgenosis chimeric embryo.
4, the transgenosis buffalo produces: the transgenic positive embryo transfer that will produce through fertilization female pronucleus injection is to spontaneous estrus or handle the same period in the replace-conceive cow intrauterine of oestrusing, and a cow is transplanted 2 pieces of embryos.B ultrasonic is observed pregnant situation after 2 months, and pregnant buffalo was continued tracing observation 10 months.
The substantial effect that the present invention gives prominence to:
Adopt the present invention to obtain high titre slow virus particle, the concentrating virus average titer reaches 10
9More than.With virus stock solution used direct infection buffalo inoblast, the positive cell infection rate can reach more than 30%.Employing zygote procaryotic injection method is expelled to concentrating virus under the buffalo mature oocyte zona pellucida or in the IVF zygote, 24% above transgenic embryos was grown to the blastaea stage, 40% above blastaea expression alien gene.Success obtains a transgenosis buffalo that changes green fluorescent protein GFP gene.
Description of drawings
Fig. 1 is the fibroblastic visible light collection of illustrative plates of buffalo with the slow virus infection of the inventive method acquisition.
Fig. 2 is the fibroblastic UV-light collection of illustrative plates of buffalo with the slow virus infection of the inventive method acquisition.
Fig. 3 is the visible light collection of illustrative plates with the buffalo embryo of the slow virus infection of the inventive method acquisition.
Fig. 4 is the UV-light collection of illustrative plates with the buffalo embryo of the slow virus infection of the inventive method acquisition.
Fig. 5 is the transgenosis buffalo pictorial diagram that mediates with the slow virus that the inventive method obtains.
Specific embodiments
By the following examples technical scheme of the present invention is described further.
The method of lentiviral vectors production transgenosis buffalo of the present invention comprises the steps:
1, high titre slow virus packing:
The go down to posterity 293T cell cultivated and 3 plasmid packaging systems and calcium phosphate transfection method of employing carries out the slow virus packing.During transfection, be that 15 μ g vector plasmids, 10 μ g envelope protein plasmid p Δ 8.9,5 μ g packaging plasmid pVSV-G and 125 μ LCaCL2 mix in test tube, supply volume to 500 μ L 3 plasmids.With 500 μ L phosphoric acid disodium hydrogens, pH value is that 6.95 TS conversion fluid is added dropwise in the above-mentioned mixed solution, and room temperature leaves standstill 30min.The plasmid mixed solution dropwise is added in the 293T cell, is 5%CO in volume percent
237 ℃ of incubators in cultivate, changed nutrient solution in second day.48～72h collects virus after the transfection.With the centrifugal 10min of supernatant 1500rpm that collects, 0.45 μ m filter filters collects virus stock solution used.Viral supernatant with 20000rpm centrifugal force, 4 ℃ of centrifugal 2h, is obtained concentrating slow virus ,-80 ℃ of freezing preservations with the PBS of 100 μ L is resuspended then.Concentrate the back virus titer after testing and reach 1 * 10
9IU.
2 slow virus infection buffalo inoblasts: the buffalo inoblast of cultivating for 4～5 generations of going down to posterity infects with slow virus stoste.During cells infected,, add 0～10 μ g/mlpolybrene except that with DMEM and 10% foetal calf serum.Transfection the 2nd day is changed normal nutrient solution and is continued culturing cell and go down to posterity 3 times, detects green fluorescent protein GFP expression under fluorescent microscope, obtains infection rate and reaches transgenic cell more than 30%.
3, slow virus infection buffalo pre-implantation embryos: adopt micro-injection method will concentrate slow virus and be injected under the buffalo mature oocyte zona pellucida or in the IVF zygote.
Buffalo Oocyte in Vitro fertilization IVF: collect the buffalo ovary from local slaughterhouse, be contained in 30 ℃ of physiological saline vacuum flask and send the laboratory back in the 4h.Adopt the 10mL syringe of No. 12 syringe needles to extract cumulus cell-ovocyte complex body COCs, under stereoscopic microscope, with tenuigenin evenly and have COCs complete or the fine and close cumulus cell of part to pick, put into the 30 * 10mm glass dish that contains 1.5mL oocyte in vitro maturation liquid and cultivate 22～24h.The ovocyte of maturation in vitro carries out IVF in vitro fertilization.
The crack virus microinjection of embryo's ovum week: test used virus injection bore and be 5～6 μ m, the locking pin bore is 20 μ m, available from U.S. HUMAGEN company, catalog number MIC-35-35.Get a 60mm culture dish lid, the CCM droplet in that 2 10 μ L are in the central authorities of ware lid with too small of paraffin oil cap, prevents the CCM evaporation.Wherein a CCM sops up, and replaces with the concentrating virus for preparing; Other one is added dropwise to ivf zygote.At first draw a certain amount of virus with syringe, be transferred to ovocyte then and drip, hold the buffalo embryo of an external after fertilization, its polar body is placed as the clock position at 6 o'clock with locking pin, from 3 o ' clock position inserting needles, viral liquid is slowly injected embryo's ovum week crack then.After finishing a batch operation, wash the embryo 2 times, put back to the vitro culture of carrying out the embryo in the incubator with CM liquid.
Embryo's vitro culture and transplanting: the zygote behind the injecting virus places the individual layer droplet with the preparation of ox granulosa cell, is 5% CO at 38.5 ℃, volume percent
2Cultivated in the incubator 7～9 days.CM liquid is changed once every 48h, checks division rate behind the cultivation 24h, and fluorescent microscope is checked the expression of EGFP in the embryo down, and the record different developmental phases is expressed embryo's number of EGFP.The blastocyst rate that record is grown after 7 days.With microscopy male blastaea move into estrus synchronization replace-conceive cow intrauterine.
(4) the transgenosis buffalo produces: the transgenic positive embryo transfer that will produce through fertilization female pronucleus injection is to spontaneous estrus or handle the same period in the replace-conceive cow intrauterine of oestrusing, and a cow is transplanted 2 pieces of embryos.B ultrasonic is observed pregnant situation after 2 months, and pregnant buffalo was continued tracing observation 10 months.
Embodiment 1
In the period of 2009 to 2010, the employing promotor is that the slow virus of CMV infects buffalo IVF zygote, has injected 582 pieces of zygotes respectively, grow to the blastaea stage for 154 pieces, account for the embryo's that annotates 26.5%, wherein 66 pieces of blastaeas are expressed GFP, account for 42.9% of blastaea sum.Get 8 pieces of positive blastaeas and be transplanted to 4 replace-conceive cow intrauterine, 2 replace-conceive cows of ultrasound diagnosis gestation after two months, wherein in birth a calf on December 17th, 2010, PCR detects the umbilical cord genome DNA sample, expression alien gene GFP and slow virus Expression element gene prove effectively production transgenosis of buffalo zygote ovum week crack injecting lentivirus buffalo.
Embodiment 2
In the period of 2009 to 2010, the employing promotor is that two kinds of slow viruss of CMV, Ubc infect the buffalo IVF zygote of different development stage respectively, injected the zygote of 466 pieces of different times respectively, grow to the blastaea stage for 113 pieces, account for the embryo's that annotates 24.2%, wherein 61 pieces of blastaeas are expressed GFP, account for 54.0% of blastaea sum.This example proof buffalo different development stage zygote ovum week crack injecting lentivirus can effectively be produced the buffalo transgenic embryos.
Claims (1)
1. method of utilizing lentiviral vectors production transgenosis buffalo, it is characterized in that: this method comprises the steps:
(1) high titre slow virus packing: 293T cell that adopting goes down to posterity cultivates and 3 plasmid packaging systems and calcium phosphate transfection method are carried out the slow virus packing; during transfection; promptly 15 μ g vector plasmids, 10 μ g envelope protein plasmid p Δ 8.9,5 μ g packaging plasmid pVSV-G and 125 μ LCaCL2 mix in test tube with 3 plasmids; supply volume to 500 μ L; with 500 μ L phosphoric acid disodium hydrogens, pH value is that 6.95 TS conversion fluid is added dropwise in the above-mentioned mixed solution; room temperature leaves standstill 30min; the plasmid mixed solution dropwise is added in the 293T cell, is 5%CO in volume percent
237 ℃ of incubators in cultivate, changed nutrient solution in second day, 48～72h collects virus after the transfection, with the centrifugal 10min of supernatant 1500rpm that collects, 0.45 μ m filter filter to be collected virus stock solution used, and viral supernatant with 20000rpm centrifugal force, 4 ℃ of centrifugal 2h, is obtained concentrated slow virus, packing with the PBS of 100 μ L is resuspended,-80 ℃ of freezing preservations concentrate the back virus titer and reach 1 * 10
9More than the IU,
(2) make buffalo transgenosis somatocyte: the buffalo inoblast of cultivating for 4～5 generations of going down to posterity infects with slow virus stoste, during cell transfecting, except that DMEM and 10% foetal calf serum, add 0～10 μ g/mlpolybrene, transfection the 2nd day is changed normal nutrient solution and is continued culturing cell and go down to posterity the green fluorescent protein GFP expression of detection cell under fluorescent microscope 3 times, the acquisition infection rate reaches the transgenosis somatocyte more than 30%
(3) make buffalo transgenosis chimeric embryo: buffalo Oocyte in Vitro after fertilization, slow virus concentrated solution 30～70pL is expelled in the buffalo IVF zygote, after cultivating 7～9 days altogether with the monolayer cell of granulosa cell is external, under fluorescent microscope, detect blastaea GFP expression, screening obtains buffalo transgenosis chimeric embryo, 24% above transgenic embryos was grown to the blastaea stage, 40% above blastaea expression alien gene
(4) the transgenosis buffalo produces: the transgenic positive embryo transfer that will produce through fertilization female pronucleus injection is to spontaneous estrus or handle the same period in the replace-conceive cow intrauterine of oestrusing, a cow is transplanted 2 pieces of embryos, B ultrasonic is observed pregnant situation after 2 months, and pregnant buffalo was continued tracing observation 10 months.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533856A (en) * | 2012-01-11 | 2012-07-04 | 广西大学 | Method for giving birth to transgenic buffalos by injecting deoxyribonucleic acids (DNA) into fertilized ovum cytoplasm |
| CN103497953A (en) * | 2013-10-10 | 2014-01-08 | 广西大学 | ShRNA (short hairpin ribonucleic acid) capable of inhibiting expression of buffalo SUV39H1 gene, lentiviral expression vector, construction method of lentiviral expression vector and applications of shRNA |
| CN108546683A (en) * | 2018-03-23 | 2018-09-18 | 广西壮族自治区水牛研究所 | A kind of method of adenovirus mediated efficient production buffalo transgenic embryo |
-
2011
- 2011-01-24 CN CN2011100253374A patent/CN102174577A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533856A (en) * | 2012-01-11 | 2012-07-04 | 广西大学 | Method for giving birth to transgenic buffalos by injecting deoxyribonucleic acids (DNA) into fertilized ovum cytoplasm |
| CN103497953A (en) * | 2013-10-10 | 2014-01-08 | 广西大学 | ShRNA (short hairpin ribonucleic acid) capable of inhibiting expression of buffalo SUV39H1 gene, lentiviral expression vector, construction method of lentiviral expression vector and applications of shRNA |
| CN103497953B (en) * | 2013-10-10 | 2015-06-03 | 广西大学 | ShRNA (short hairpin ribonucleic acid) capable of inhibiting expression of buffalo SUV39H1 gene, lentiviral expression vector, construction method of lentiviral expression vector and applications of shRNA |
| CN108546683A (en) * | 2018-03-23 | 2018-09-18 | 广西壮族自治区水牛研究所 | A kind of method of adenovirus mediated efficient production buffalo transgenic embryo |
| CN108546683B (en) * | 2018-03-23 | 2021-09-21 | 广西壮族自治区水牛研究所 | Method for efficiently producing transgenic buffalo embryos by adenovirus mediation |
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Application publication date: 20110907 |