CN101974565A

CN101974565A - Method for producing transgenic buffalo embryos by applying intracytoplasmic sperm injection (ICSI) mediation

Info

Publication number: CN101974565A
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: 2011-02-16
Anticipated expiration: 2030-08-30
Also published as: CN101974565B

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 the southern a kind of important domestic animal that has DEVELOPMENT PROSPECT of China,, high-temp resisting high-humidity resisting strong because of its adaptability, disease resistance are strong, crude feed tolerance, raise easily, working life is long and characteristic such as dairy products nutritive value height, and extremely people pay close attention to and pay attention to.Yet the 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 mediation transgenosis (ICSI-Mediated Gene Transfer, ICSI-Tr) technology, hatch by preparing highly purified transgene carrier and sperm, carry out monosperm microinjection fertilization in the kytoplasm then, produce transgenic embryos, the expression of combined with fluorescent microscopic examination marker gene EGFP (Enhanced Green Fluorecence Protein) filters out transgenic embryos and transplants, and then obtains transgenic animal.That the ICSI transgenic technology has is simple, good reproducibility, efficient height, can carry out advantages such as big fragment gene (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 seminal fluid 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.Draw the sperm on upper strata then, 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 its bonding force foreign DNA.Be that the linearizing transgene carrier pEGFP-N1 of 2-10 μ mg/mL was hatched 5 minutes altogether with concentration then, be added to and contain the micrurgy liquid (MM that weightmeasurement ratio is 7% polyvinylpyrrolidone (PVP), TCM199+5mmol/LNaHCO3+10mmol/L Hepes) in, prepares the ICSI operation.

2. microinjection in the buffalo sperm kytoplasm: hold an external sophisticated buffalo ovocyte with fixing needle tubing, use intracytoplasmic sperm injection needle tubing inserting needle, pumpback endochylema then, when confirming that the archiblast film stops pumpback at once when broken and also kytoplasm and sperm slowly injected in the ovum, inject PVP less as far as possible.Subsequently, withdraw from syringe soon 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 handles 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 handling 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 behind the cultivation 24h, the blastocyst rate that record is grown after 7 days.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.In the wherein 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.It is intracytoplasmic sperm injection (200 *) in the ooecium matter.

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

Fig. 3. the micro-image of the buffalo embryo of the early stage spilting of an egg 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 ovocyte: the buffalo ovary is collected in local slaughterhouse from the Nanning City, is contained in 25～37 ℃ and contain K ⁺, Ca ²⁺, Mg ²⁺The physiological saline vacuum flask in deliver to the laboratory in the 4h.Ovary is cut ligamentum suspensorium ovarii with scissors after cleaning 3 times with 37 ℃ physiological saline.Extracting earlier the ovary surface diameter with the 10mL syringe that has No. 12 syringe needles 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), 60mg/L penicillin, 100mg/L 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: behind the buffalo semen thawing, adopt suspension method to separate the sperm of high vigor, (sperm adds the DPBS 0.5mL that contains 1%TritonX-100 to adopt freeze-thaw method (multigelation in liquid nitrogen) or TritonX-100 processing then, 4 ℃ of static 5 minutes after scouring are 2 times behind the mixing) or ultrasonication (get 1mL upstream sperm in a test tube, with ultrasonication 3 minutes, wash then 2 times).

Sperm and carrier DNA are hatched altogether: application carrier of the present invention 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, after electrophoresis detection observation enzyme cuts entirely, use alcohol precipitation and carry out the purifying recovery, the ultrapure water dissolving, measure concentration, be distributed into the every pipe of 10uL ,-20 ℃ of preservations are standby.The EGFP plasmid is diluted to different concentration according to the experiment needs, at room temperature hatches and be added in the micrurgy liquid that contains 7%PVP after 5 minutes, prepare the ICSI operation with the sperm of handling well.

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, and available from U.S. HUMAGEN company (MIC-35-35), ICSI is pre-installed standby on the micrurgy instrument.Get the lid of a Nunc 60mm culture dish, do the micrurgy liquid droplet of several 15 μ L as required, use too small of paraffin oil cap then, prevent the evaporation of micrurgy liquid in the central authorities of lid.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, be transferred to the micrurgy drop of placing ovocyte then, hold an external sophisticated buffalo ovocyte with locking pin, put its first polar body in as the clock position at 6 o'clock, then from 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 the acceleration flow state suddenly, endochylema confirms that the archiblast film is broken, stop pumpback at once kytoplasm and sperm are slowly injected in the ovum, inject PVP as far as possible less.Subsequently, withdraw from syringe soon 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 handles 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 handling 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 behind the cultivation 24h, the blastocyst rate that record is grown after 7 days.The embryo placed under the fluorescent microscope check the expression of EGFP the embryo.The record different developmental phases is expressed embryo's number of EGFP.

Embodiment 1

In the period of 2008 to 2009, in the wherein 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

In the period of 2008 to 2009, we inject 95 ovocytes, activate back 86 division (accounting for 90.5%) has taken place, and have wherein expressed EGFP (accounting for 41.9%) for 36, vitro culture obtains 16 pieces of blastaeas after 7 days, wherein expressed EGFP (accounting 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:

(1) hatching altogether of buffalo sperm and transgenosis DNA: the buffalo seminal fluid 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 its bonding force foreign DNA; Be that the linearizing transgene carrier pEGFP-N1 of 2-10 μ mg/mL was hatched 5 minutes altogether with concentration then, be added to and contain in the micrurgy liquid that weightmeasurement ratio is 7% polyvinylpyrrolidone, prepare the ICSI operation;

(2) microinjection in the buffalo sperm kytoplasm: hold an external sophisticated buffalo ovocyte with fixing needle tubing, use intracytoplasmic sperm injection needle tubing inserting needle, pumpback endochylema then, when confirming that the archiblast film stops pumpback at once and slowly injects in the ovum when broken to kytoplasm and sperm; Subsequently, withdraw from syringe soon 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 handle 5min, cultivate 5h with the cycloheximide that contains 10 μ g/mL then, clean 2 times with the 1mL nutrient solution again, move in the culture dish and cultivate;

(3) cultivation of transgenosis buffalo embryo and evaluation: the ICSI ovocyte that activates after handling 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 behind the cultivation 24h, the blastocyst rate that record is grown after 7 days; The blastaea that obtains is detected the GFP expression under fluorescent microscope, screening obtains the transgenosis buffalo embryo.