CN101974565B

CN101974565B - Method for producing transgenic buffalo embryos by applying intracytoplasmic sperm injection (ICSI) mediation

Info

Publication number: CN101974565B
Application number: CN 201010266475
Authority: CN
Inventors: 石德顺; 陈自红; 刘庆友
Original assignee: Guangxi University
Current assignee: Guangxi University
Priority date: 2010-08-30
Filing date: 2010-08-30
Publication date: 2013-04-24
Anticipated expiration: 2030-08-30
Also published as: CN101974565A

Abstract

The invention relates to a method for producing transgenic buffalo embryos by applying intracytoplasmic sperm injection (ICSI) mediation. The method comprises the following steps of: 1, incubating buffalo sperms and transgenic deoxyribonucleic acid (DNA) together; 2, performing intracytoplasmic microinjection on the buffalo sperms; and 3, cultivating and identifying the transgenic buffalo embryos. By the method of the invention, the transgenic buffalo embryos are obtained successfully. In 102 injection spawns of the transgenic buffalo embryos, 61.8 percent of the injection spawns are subjected to cleavage, 17 blastospheres are obtained in embryos expressed by blastomere serving as enhanced green fluorescent protein (EGFP), 11 blastospheres express the EGFP, and the transgenic positive rate of the blastospheres is up to 64.7 percent; and after one of the transgenic embryos is cultured in vitro for a long time, the expression of the EGFP can be still seen during 2 to 7 days, so the method makes the important progress for producing transgenic buffalos further.

Description

Use the method for ICSI mediation production transgenosis buffalo embryo

Technical field

The invention belongs to engineered transgenic animal technology, particularly use the method for ICSI mediation production transgenosis buffalo embryo.

Background technology

Buffalo is a kind of important domestic animal that has DEVELOPMENT PROSPECT of south China, because of its strong adaptability, high-temp resisting high-humidity resisting, disease resistance are strong, crude feed tolerance, raise easily, working life is long and the characteristic such as dairy products nutritive value height, and extremely people pay close attention to and pay attention to.Yet milk yield and the breeding potential of the kind of China buffalo are low at present, seriously restrict the development of buffalo milk industry.ICSI gene transfer (ICSI-Mediated Gene Transfer, ICSI-Tr) technology, hatch by preparing highly purified transgene carrier and sperm, then carry out monosperm microinjection fertilization in the kytoplasm, produce transgenic embryos, combined with fluorescent microscopic examination marker gene EGFP(Enhanced Green Fluorecence Protein) expression filters out transgenic embryos and transplants, and then obtains transgenic animal.That the ICSI transgenic technology has is simple, good reproducibility, efficient are high, can carry out the advantage such as large fragment gene (such as artificial chromosome) transfer.Therefore, utilize the ICSI transgenic technology to solve the technological method that the milk yield of the existing buffalo kind of China and the low problem of breeding potential will be a kind of feasible and effect stabilities.

Summary of the invention

The technical problem to be solved in the present invention provides a kind of method of the ICSI of application mediation production transgenosis buffalo embryo.

The present invention solves the problems of the technologies described above with following technical scheme:

1. hatching altogether of buffalo sperm and transgenosis DNA: the Buffalo Semen that will thaw place contain 1mL be subjected to seminal fluid (the platform Luo Shi liquid of improvement suspended in test tube mTALP) 30～60 minutes.Then draw the sperm on upper strata, and twice of eccentric cleaning (centrifugal force 300g).Then, take freeze-thaw method or TritonX100 or supersonic method to destroy the plasma membrane of sperm, increase it to the bonding force of foreign DNA.Then be that the linearizing transgene carrier pEGFP-N1 of 2-10 μ g/mL was hatched 5 minutes altogether with concentration, be added to and contain the micrurgy liquid (MM that weightmeasurement ratio is 7% polyvinylpyrrolidone (PVP), TCM199+5mmol/L NaHCO3+10mmol/L Hepes) in, prepares the ICSI operation.

2. microinjection in the buffalo sperm kytoplasm: the buffalo oocytes that holds an external maturation with fixing needle tubing, then use intracytoplasmic sperm injection needle tubing inserting needle, pumpback endochylema, when confirming that the archiblast film stops at once pumpback when broken and also kytoplasm and sperm slowly injected in the ovum, inject less PVP as far as possible.Subsequently, withdraw from soon syringe after elder generation is light, and ovocyte is discharged from fixed tube.The ovocyte of finishing ICSI is cultivated 1h in incubator after, with containing 5 μ mol/L ionomycin (Ionomycin, ION) nutrient solution (TCM199 add 5% send out clear bovine serum (OCS)) activates processes 5min, then with the cycloheximide (Cycloheximide that contains 10 μ g/mL, CHX) cultivate 5h, clean 2 times with the 1mL nutrient solution afterwards, move in the culture dish and cultivate.

3. the cultivation of transgenosis buffalo embryo and evaluation: the ICSI ovocyte that activates after processing places the individual layer droplet for preparing with the ox granulosa cell, at 38.5 ℃, 5%CO ₂Cultivated in the incubator of maximal humidity 7～9 days.Nutrient solution is changed once every 48h, checks division rate after cultivating 24h, 7 days rear record blastocyst rates of growing.The blastaea that obtains is detected the GFP expression under fluorescent microscope, screening obtains the transgenosis buffalo embryo.

Use method of the present invention, successfully obtained the transgenosis buffalo embryo.Wherein in a collection of 102 injection ovum, 63 divisions (accounting for 61.8%) are arranged, 38 of the embryos (accounting for 60.3%) that blastomere EGFP expresses, 17 blastaeas have been obtained, wherein 11 blastaeas have been expressed EGFP, and the transgenic positive rate of blastaea has been carried out long vitro culture up to 64.7% to one of them transgenic embryos, in the time of 27 days, still can see the expression of EGFP, for next step production transgenosis buffalo has stepped an important step.

Description of drawings

Fig. 1. the micro-image of injection buffalo sperm in the ovocyte kytoplasm of the present invention.Be ovum intracytoplasmic sperm injection (200 *).

Fig. 2. 1 cell stage embryo's of 2 polar bodys micro-image is discharged in the activation that the present invention obtains afterwards.Be that EGFP expresses (200 *) in the ICSI ovum

Fig. 3. the micro-image of the buffalo embryo of the Early cleavage that the present invention obtains.Be that EGFP is in ICSI embryo cleavage stage expression (200 *)

Fig. 4. the morular micro-image of buffalo of the expression EGFP that the present invention obtains.Be that EGFP is in ICSI embryo morula expression (200 *)

Fig. 5. the micro-image of the buffalo blastaea of the expression EGFP that the present invention obtains.Be EGFP in that the ICSI embryo expresses (200 *) blastula stage

Fig. 6. the micro-image of the buffalo hatched blastocyst inner cell mass of the expression EGFP that the present invention obtains.Be that EGFP is hatched expression (200 *) in the blastaea at ICSI

Embodiment

The present invention has used genetic engineering technique, feasibility and the influence factor of buffalo ICSI-Tr have been carried out system's further investigation, has set up the technological method of the stable production buffalo transgenic embryos of a cover.The concrete steps of present technique are:

The preparation of buffalo oocytes: buffalo ovaries is collected in local slaughterhouse from the Nanning City, is contained in 25～37 ℃ and contain K ⁺, Ca ²⁺, Mg ²⁺The physiological saline heat preservation bottle in deliver to the laboratory in the 4h.Ovary is cut ligamentum suspensorium ovarii with scissors for 3 times afterwards with 37 ℃ physiological saline cleaning.Using 10mL syringe with No. 12 syringe needles to extract first the ovary surface diameter is ovocyte in 2～6mm ovarian follicle.Then, it is even to select tenuigenin at stereoscopic microscope, and ovocyte complete or the fine and close cumulus cell of part is arranged, and puts into to contain 1.5mL oocyte in vitro maturation liquid (composition is TCM199,5.0mmol/L Hepes, 26.2mmol/LNaHCO ₃, 5% OCS, 0.1 μ g/mL follitropin (FSH), 60mgL penicillin, 100mgL Streptomycin sulphate) glass dish (in 30 * 10mm), place 38.5 ℃, 5%CO ₂With cultivate 22～24h in the incubator of maximum saturation humidity.

The preparation of buffalo sperm: after Buffalo Semen thaws, adopt suspension method to separate the sperm of high vigor, then (sperm adds the DPBS 0.5mL that contains 1%TritonX-100 to adopt freeze-thaw method (multigelation in liquid nitrogen) or TritonX-100 processing, 4 ℃ of washings after static 5 minutes are 2 times behind the mixing) or ultrasonication (get 1mL upstream sperm in a test tube, with ultrasonication 3 minutes, then wash 2 times).

Sperm and carrier DNA are hatched altogether: the carrier that the present invention uses is pEGFP-N1, contains two goal gene of EGFP and neo.The purifying of carrier DNA goes the operation instructions (Cat.12362) of intracellular toxin plasmid extraction test kit to carry out by QIAGEN company.The plasmid pEGFP-N1 of purifying cuts and after PCR identifies, cuts 3 ~ 4h with 37 ℃ of enzymes of ApalI enzyme through enzyme, electrophoresis detection observe enzyme cut complete after, use alcohol precipitation and carry out purifying and reclaim, the ultrapure water dissolving, mensuration concentration is distributed into the every pipe of 10uL ,-20 ℃ save backup.The EGFP plasmid is diluted to different concentration according to the experiment needs, at room temperature hatches with the sperm of handling well and be added in the micrurgy liquid that contains 7%PVP after 5 minutes, prepare the ICSI operation.

Injection in the ooecium matter of buffalo sperm: this is tested used intracytoplasmic sperm injection needle tubing internal diameter and is 9～10 μ m, and the locking pin bore is 20 μ m, available from U.S. HUMAGEN company (MIC-35-35), is contained on the micrurgy instrument for subsequent use before the ICSI.Get the lid of a Nunc 60mm culture dish, do as required the micrurgy liquid droplet of several 15 μ L in the central authorities of lid, then use too small of paraffin oil cap, prevent the evaporation of micrurgy liquid.Micrurgy liquid is wherein sopped up, drip replacement with the sperm of mixing PVP.Add 20 ready ovum at other 1 micrurgy liquid and begin the ICSI micrurgy.With syringe sperm drip the bottom along sperm tail with its tail pipe in, then be transferred to the micrurgy drop of placing ovocyte, hold the buffalo oocytes of an external maturation with locking pin, put its first polar body in such as the clock position at 6 o'clock, then from such as 3 o'clock of clock inserting needle, go deep into about 2/3 beginning pumpback endochylema in the ooecium matter perpendicular to polar body, when presenting suddenly the acceleration flow state, endochylema confirms that the archiblast film is broken, stop at once pumpback kytoplasm and sperm are slowly injected in the ovum, inject less PVP as far as possible.Subsequently, withdraw from soon syringe after elder generation is light, and ovocyte is discharged from fixed tube.After finishing a batch operation, the ICSI ovocyte is cleaned 2 times with nutrient solution, in incubator, cultivate behind the 1h with containing 5 μ mol/L ionomycin (Ionomycin, ION) nutrient solution activates processes 5min, then with containing 10 μ g/mL cycloheximide (Cycloheximide, CHX) nutrient solution is cultivated 5h, cleans 2 times with 1mL CM afterwards, moves in the culture dish and cultivates.

ICSI-Tr embryo's vitro culture and evaluation: the ICSI ovocyte that activates after processing places the droplet that contains ox granulosa cell monolayer cell, at 38.5 ℃, 5%CO ₂Cultivated in the incubator of maximal humidity 7～9 days.Nutrient solution is changed once every 48h, checks division rate after cultivating 24h, 7 days rear record blastocyst rates of growing.The embryo placed under the fluorescent microscope check that EGFP is at embryo's expression.The record different developmental phases is expressed embryo's number of EGFP.

Embodiment 1

Between 2008 to 2009, wherein in a collection of 102 injection ovum, 63 divisions (accounting for 61.8%) are arranged, and 38 of the embryos (accounting for 60.3%) that blastomere EGFP expresses have obtained 17 blastaeas, wherein 11 blastaeas have been expressed EGFP, the transgenic positive rate of blastaea has been carried out long vitro culture up to 64.7% to one of them transgenic embryos, still can see the expression of EGFP in the time of 27 days, the proof adopting said method, effectively production transgenosis buffalo embryo.

Embodiment 2

Between 2008 to 2009, we inject 95 ovocytes, activate rear 86 division (accounting for 90.5%) has occured, and have wherein expressed EGFP(and account for 41.9% for 36), vitro culture obtains 16 pieces of blastaeas after 7 days, wherein expressed EGFP(and account for 62.5% for 10 pieces).This embodiment proves, the method for the method production transgenosis buffalo embryo by the ICST mediation of the present invention is feasible.

Claims

1. use the method that ICSI mediates production transgenosis buffalo embryo for one kind, it is characterized in that processing step is:

⑴ buffalo sperm and transgenosis DNA's hatches altogether: the Buffalo Semen that will thaw is drawn the sperm on upper strata after placing and containing test tube that 1mL is subjected to seminal fluid and suspended 30～60 minutes, and twice of eccentric cleaning; Then, take freeze-thaw method or TritonX100 or supersonic method to destroy the plasma membrane of sperm, increase it to the bonding force of foreign DNA; Then be that the linearizing transgene carrier pEGFP-N1 of 2-10 μ g/mL was hatched 5 minutes altogether with concentration, be added to and contain in the micrurgy liquid that weightmeasurement ratio is 7% polyvinylpyrrolidone, prepare the ICSI operation;

⑵ microinjection in the buffalo sperm kytoplasm: the buffalo oocytes that holds an external maturation with fixing needle tubing, then use intracytoplasmic sperm injection needle tubing inserting needle, pumpback endochylema, when confirming that the archiblast film stops at once pumpback and slowly injects in the ovum when broken to kytoplasm and sperm; Subsequently, withdraw from soon syringe after elder generation is light, and ovocyte is discharged from fixed tube; The ovocyte of finishing ICSI is cultivated 1h in incubator after, activate with the nutrient solution that contains 5 μ mol/L ionomycins and to process 5min, then cultivate 5h with the cycloheximide that contains 10 μ g/mL, clean 2 times with the 1mL nutrient solution again, move in the culture dish and cultivate;

⑶ cultivation and the evaluation of transgenosis buffalo embryo: the ICSI ovocyte that activates after processing places the individual layer droplet for preparing with the ox granulosa cell, at 38.5 ℃, 5%CO ₂Cultivated in the incubator of maximal humidity 7～9 days; Nutrient solution is changed once every 48h, checks division rate after cultivating 24h, 7 days rear record blastocyst rates of growing; The blastaea that obtains is detected the GFP expression under fluorescent microscope, screening obtains the transgenosis buffalo embryo.