CN101654687A - Method for preparing transgenic pig by hatching slow viruses and sperms - Google Patents
Method for preparing transgenic pig by hatching slow viruses and sperms Download PDFInfo
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- CN101654687A CN101654687A CN200910192144A CN200910192144A CN101654687A CN 101654687 A CN101654687 A CN 101654687A CN 200910192144 A CN200910192144 A CN 200910192144A CN 200910192144 A CN200910192144 A CN 200910192144A CN 101654687 A CN101654687 A CN 101654687A
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Abstract
The invention discloses a method for preparing a transgenic pig by hatching slow viruses and sperms, comprising the following steps: enabling the sperms to absorb slow virus vectors after the slow virus vectors and the sperms are co-incubated; then inseminating female animals, and screening the transgenic pig from offspring animals. The invention adopts a co-incubation technique of the slow viruses and the sperms, guides the slow viruses into oosperms in such a way that the sperms absorb the slow viruses and then is inseminated or fertilized in vitro and integrates foreign genes into an embryonic genome so as to prepare the transgenic pig. the invention solves the problems of the very low integration efficiency, the unstable heredity, and the like of the transgenic pig which is mediated insuch a way that the sperms absorb naked DNA. The method has the outstanding advantage of combining the respective advantages of the sperms and the slow virus vectors, easy and convenient operation and lower cost; in addition, the method utilizes the slow virus vectors and the sperms to hatch and is a transgenic method with low cost, easy and convenient operation and higher transgenic efficiency.
Description
Technical field
The present invention relates to prepare the new technology of transgenic pig.
Background technology
Sperm mediated gene transfer method is to obtain simple and one of the high-efficiency method of transgenic pig at present.This method had successful report on animals such as mouse, pig, but in the research subsequently, because genetically modified efficient is low and once controversial, and different laboratory results reported is widely different, has the inefficient problem of exogenous origin gene integrator.
Slow virus is considered to prepare the novel excellent genes transfer vector of transgenic pig.Slow virus can infect somatoblast can infect Unseparated Cell again, can be from the sexual cell to the blastula stage each etap foreign gene transfer.The animals such as production transgenic mouse, sheep, ox, pig, chicken, fish that utilized the success of lentiviral vectors infection animal embryo, zygote and stem spermatogonium at present.
Studies confirm that in a large number sperm and slow virus are the ideal carriers of transgenosis, but relevant slow virus and sperm are hatched the research for preparing transgenic pig altogether and are not appeared in the newspapers both at home and abroad.
Summary of the invention
The purpose of this invention is to provide a kind of slow virus and sperm and hatched the method for preparing transgenic pig.
A kind of slow virus and sperm are hatched the method for preparing transgenic pig, comprise the steps: that lentiviral vectors and sperm are hatched altogether after, make sperm absorption lentiviral vectors; Then through the artificial insemination jenny, at filial generation animal screening transgenic pig.
A kind of slow virus and sperm are hatched the method for preparing transgenic pig, comprise the steps: that lentiviral vectors and sperm are hatched altogether after, make sperm absorption lentiviral vectors; Through in vitro fertilization, embryo transfer after the vitro culture obtains transgenic pig.
Hatch in the method for preparing transgenic pig above-mentioned slow virus and sperm, the method that described slow virus and sperm are hatched altogether is: animal semen diluent flush away seminal plasma, the sperm of getting the flush away seminal plasma is in the artificial insemination bottle, add slow virus liquid, lid tightly is put in 17 ℃ of insulation can 120min, and every therebetween 15min stirs the seminal fluid bottle gently, prevents the gathering of sperm precipitation, kind of the preceding 10min that provides and delivers checks a vigor, and its vigor reaches more than 0.5 can carry out artificial insemination.
Hatch in the method for preparing transgenic pig above-mentioned slow virus and sperm, described test-tube method is: select the Healthy female animal, and in intramuscular injection on the same day human villin gonadal hormone of oestrusing, the laggard pedestrian worker's semen deposition of intramuscular injection.
Compared with prior art, the present invention has following beneficial effect:
The technology that the present invention adopts slow virus and sperm to hatch altogether by artificial insemination or in vitro fertilization again behind the sperm absorption slow virus, imports slow virus in the zygote, and with exogenous origin gene integrator in the embryonic gene group, thereby the preparation transgenic pig.Thereby problems such as sperm absorption naked DNA mediation transgenic pig integration rate is very low, hereditary instability have been overcome.The outstanding feature of this method is the advantage separately that combines slow virus and sperm vector, and is easy and simple to handle, cost is lower.Utilizing lentiviral vectors and sperm to hatch, is a kind of low cost, easy and simple to handle, transgenic method that transgene efficiency is higher.
Description of drawings
Fig. 1 is transgenosis piglet eGFP Fluirescence observation figure.
Fig. 2 detects transgenosis piglet electrophorogram for artificial insemination PCR.
1-8 is the piglet sample; Remove 3 extras, other all can detect the existence of gene.
The Southern blot that Fig. 3 detects positive transgenosis piglet for artificial insemination PCR is figure as a result.Control is known positive findings, positive positive piglet results of hybridization.
Embodiment
Embodiment 1 lentiviral vectors packing
Prepare lentiviral vectors with the 293T packing cell.24h before plasmid DNA cotransfection 293T cell, had digestive transfer culture 293T cell, inoculation 6 * 10
6Individual cell.Preceding 2～the 3h of plasmid transfection changes cell culture medium, adds fresh culture.Calculate the requirement (packaging plasmid 6125 μ g, transferring plasmid 6125 μ g and adventitia plasmid 2150 μ g) of every kind of plasmid according to the quantity of the concentration of plasmid DNA and transfectional cell.After cultivating 8h, change fresh culture.After transfection the the 1st, the 2nd and the 3rd day taked to change fully fresh medium and do not changed the nutrient solution dual mode and collect the preparation virus vector respectively, and measures the carrier titre.The virus vector of results different batches preparation, after a large amount of preparation of employing test kits filtered, the high speed gradient centrifugation is collected, and was standby in-80 ℃ of preservations.
The preparation of embodiment 2 transgenic pigs
1, pig sperm and lentiviral vectors hatches altogether
Diluting boar semen liquid (Swine Fertilization Medium, SFM) preparation: 11.25g dextrose anhydrous 10g citrate dihydrate trisodium 4.7g two water disodium ethylene diamine tetraacetate 3.25g Citric acid monohydrate Food grade 6.5g Tutofusin triss add 1 liter of distilled water constant volume, add 6g bovine serum albumin (BSA) at last, now with the current.
Determine (1) and join boar that the check semen quality writes down qualified order vigor three times and reaches more than 0.7 recently.Select test to adopt the highest that part seminal fluid of motility of sperm in the landrace seminal fluid same day, collect in the seminal fluid dense thick part 5mL in the 15mL centrifuge tube, add the SFM/BSA liquid 5mL that prepared also preheating the same day (note the temperature difference must less than 1 ℃) between the two.Room temperature is placed 5min.
(2) above-mentioned seminal fluid is transferred in the 50ml centrifuge tube that 40ml SFM/BSA is housed, 25 ℃, the careful suction of the centrifugal 10min of 800g removed supernatant, add 40ml SFM/BSA (25 ℃) spermatozoa diluent and remove supernatant in careful suction of 17 ℃ of centrifugal 10min of 800g, add 3-4ml SFM/BSA (17 ℃) and blow and beat gently, make sperm resuspended.
(3) detect motility of sperm, and counting, reach more than 0.65 promptly desirable 10 as motility of sperm
9Individual sperm is in the artificial insemination bottle of 40ml SFM/BSA (17 ℃), add 50ug DNA, suction is defoamed, lid tightly is put in 17 ℃ of insulation can 100min, every therebetween 15min stirs the seminal fluid bottle gently, prevent the gathering of sperm precipitation, kind of the preceding 10min check vigor of providing and delivering, its vigor reaches more than 0.5 can carry out artificial insemination.
2, the preparation of transgenic pig
(1) artificial insemination: in intramuscular injection on the same day human villin gonadal hormone (HCG) of oestrusing.Behind the intramuscular injection HCG 24h, carry out the artificial insemination, every side 1mL (contains sperm 5 * 10 approximately
7Individual/mL).Divide the puerperium, identify the transgenic positive piglet with PCR, Southern blot.PCR the results are shown in Figure 2, and Southern blot the results are shown in Figure 3.The PCR method is a kind of succinct authentication method fast, owing to there is certain false positive, is used for the detection of preliminary positive piglet.Southern blot is a kind of positive identification method of classics, and the accuracy rate height is used for further positive detection, and both results have obtained success in conjunction with the explanation transgenosis.
(2) in vitro fertilization: as cumulus cell and the ovocyte complex body (COCs) of vitro culture 42-44h to be taken out, take off after the cumulus cell, cushion nutrient solution (mTBM) washing 3 times with TRIS; Place 25 sophisticated ovocytes in each 50 μ LmTBM drop; In incubator, cultivate after the 30-45min, add the seminal fluid that 50 μ L handle well before this in each drop, add the lentiviral vectors of high titre simultaneously; With sperm, ovocyte and high titre lentiviral vectors mixed solution in incubator, 39 ℃, 5%CO
2Air, saturated humidity is cultivated 7h; With possible zygote with embryo medium washing 3 times after, according to 20-25 embryo/50 μ L, 39 ℃, 5%CO
2Air, saturated humidity is cultivated; Calculating spilting of an egg rate and blastaea rate and positive embryo at 2d and 7d respectively leads.Adopt common oviduct transplantation, divide the puerperium, identify the transgenic positive piglet with direct viewing fluorescence, PCR, Southern blot.Fluorescence the results are shown in Figure 1, and PCR the results are shown in Figure 2, and Southernblot the results are shown in Figure 3.
Claims (4)
1. slow virus and sperm are hatched the method for preparing transgenic pig, it is characterized in that comprising the steps: that lentiviral vectors and sperm are hatched altogether after, make sperm absorption lentiviral vectors; Through the artificial insemination jenny, in the filial generation pig, screen transgenic animal then.
2. slow virus and sperm are hatched the method for preparing transgenic pig, it is characterized in that comprising the steps: that lentiviral vectors and sperm are hatched altogether after, make sperm absorption lentiviral vectors; Through in vitro fertilization, embryo transfer after the vitro culture obtains transgenic pig.
3. the method for preparing transgenic pig as claimed in claim 1 or 2, it is characterized in that the method that described slow virus and sperm are hatched altogether is: animal semen diluent flush away seminal plasma, the sperm of getting the flush away seminal plasma is in the artificial insemination bottle, add slow virus stoste, lid tightly is put in 17 ℃ of insulation can 120min, and every therebetween 15min stirs the seminal fluid bottle gently, prevents the gathering of sperm precipitation, kind of the preceding 10min that provides and delivers checks a vigor, and its vigor reaches more than 0.5 can carry out artificial insemination.
4. the method for preparing transgenic pig as claimed in claim 1 is characterized in that described test-tube method is: select the Healthy female pig, and in intramuscular injection on the same day human villin gonadal hormone of oestrusing, the laggard pedestrian worker's semen deposition of intramuscular injection.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115868447A (en) * | 2023-02-15 | 2023-03-31 | 新疆天澳牧业有限公司 | Breeding management method for synchronous estrus returning and timed semen deposition of young cattle |
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2009
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115868447A (en) * | 2023-02-15 | 2023-03-31 | 新疆天澳牧业有限公司 | Breeding management method for synchronous estrus returning and timed semen deposition of young cattle |
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Application publication date: 20100224 |