CN100480390C - Transgenic cow obtaining method - Google Patents
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- CN100480390C CN100480390C CNB2005100709752A CN200510070975A CN100480390C CN 100480390 C CN100480390 C CN 100480390C CN B2005100709752 A CNB2005100709752 A CN B2005100709752A CN 200510070975 A CN200510070975 A CN 200510070975A CN 100480390 C CN100480390 C CN 100480390C
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Abstract
The invention discloses an obtaining method of gene-transition cow, which comprises the following steps: connecting target carrier with linear beta-milk globulin protein mutation gene with somatic cell through isogenesis recombination; forming beta-milk globulin protein gene silence conduct cell; guiding the conduct cell in the cow enucleated mother cell to rebuild embryo; transplanting the recombined embryo in the pregnant cow uterus. The method makes cow produce more milk without beta-milk globulin protein, which is more nutritious for people.
Description
Technical field
The present invention relates to the preparation method of the improved transgenic cattle of a kind of milk composition in animal transgenic technology and the cell engineering field.
Background technology
" one glass of strong nationality of milk ", along with growth in the living standard, people are increasing to the demand of milk preparation.At present, China has entered the quick rise period of milk preparation demand, and the pre-capita consumption rate of increase of milk preparation all surpasses 10% every year in recent years.To 2015, Chinese dairy products total quantity consumed will reach 2,500 ten thousand tons, increase by 6.8% every year, and rate of growth is higher than the world 1-2% of the same period.But the production of Chinese dairy products also is in a relatively backward level.Along with the increase to the milk preparation demand, the quality of milk preparation more and more is subjected to people's attention.
It is undisputable fact that absorption milk can cause the human allergy.Milk allergy disease is meant that human body produces anaphylaxis to milk (mainly being some protein such as α-S1 casein and the beta-lactoglobulin in the milk), also is called milk proteins and does not tolerate.Some protein in the milk such as α-S1 casein and beta-lactoglobulin are the allergens of generally acknowledging at present.Under the normal circumstances,, these two kinds of albumen mass-energy can not cause the anaphylaxis (being that allergen can be destroyed by digestion) of human body if being absorbed by human consumption.But if the digestion ability deficiency of human body, then these two kinds of protein may enter human body with the form that is not digested, thereby cause allergic reaction.
Newborn infants and the elderly are the crowds that falls ill easily.The elderly is difficult to digestion because digestion ability descends to macromole long-chain protein, and newborn infants then is because their Digestive tract and immunity system zoon not as yet.Generally for newborn infants, 0-6 month is the most responsive period.
Tackle infant's milk allergy disease two kinds of ways are arranged: cut off allergen and pharmacological agent.
Though pharmacological agent can be controlled clinical symptom hypersensitive at short notice, but after the drug withdrawal, if baby continues edible milk, symptom then hypersensitive can occur again once more.And the side effect of pharmacological agent potential can not be ignored, and especially at fash (eczema, infantile eczema) symptom, uses the hormone ointment through regular meeting.And prolonged and repeated use hormone medicine can produce bigger detrimentally affect to baby.
The protein that contains the 2.2-4.4% that has an appointment in the normal milk is one of most important nutritive substance in the cow's milk.The ox protein of milk mainly comprises casein, whey-protein and a spot of fat globule membrane albumen etc.Beta-lactoglobulin belongs to a kind of of whey-protein, and the content in cow's milk is approximately 2-4g/L.Beta-lactoglobulin is a topmost whey-protein in the ruminating animal, also is present in some non-ruminant animal, such as pig, horse, cat, dog, kangaroo, dolphin and whale.But do not contain this albumen in the milk of the mankind, rodents and Lagomorpha.Under normal physiological conditions, beta-lactoglobulin exists with the form of homodimer, and molecular weight is 36,400 dalton.PH2-3 or pH greater than 9 condition under, can depolymerization be monomer, at pH3.5-5.5, under 4 ℃ the condition, can form eight aggressiveness, promptly 4 dimers exist with 422 symmetries.
The full length gene 4729bp of coding beta-lactoglobulin, comprise 7 exons and 6 introns, 178 amino-acid residues of its primary transcript coding, the signal peptide that comprises 16 amino-acid residues of length, its mature peptide contains 162 amino-acid residues, wherein α-Luo Xuanjiegou accounts for 15%, and the beta sheet structure accounts for 50%.
The molecular biology basis of gene knockout technology is homologous recombination (homologous recombination), and the homologous recombination of gene is more general biological phenomena, to eukaryote existence is arranged all from phage, bacterium.The homologous recombination odds is generally 10
-5-10
-7Between, but not the occurrence probability of homologous recombination but can reach 10
-2-10
-4Between.Homologous recombination is removed and can be carried out between dna molecular, also can be at RNA carries out between intermolecular or RNA and dna molecular.
The gene knockout technology is to set up in yeast saccharomyces cerevisiae (S.cerevisiae) seventies.1985, people such as Smithies utilize homologous recombination one section exogenous plasmid P Δ β 117 to be inserted into the β-globin site of human chromosome first, this is the successful first example (Smithies of practicing shooting in mammalian cell, Ronald G etc., Insertion of DNAsequences into the human chromosomal β-globin locus by homologousrecombination.Nature, 1985,317:230-234).1987, two research groups such as Thomas and Capecchi are reported in the location that ES (Embryo Stem) cell carried out gene simultaneously and knock out (Thomas K R and Capecchi etc., Site-directed mutagenesis by gene targeting in mouseembryo-derived stem cells.Cell, 1987,51:503-512).After this, the research of carrying out gene knockout with mouse ES cells is flourish, has now become research gene structure function and a kind of normal experiment method of setting up people's heredopathia model.The mutant mice of having set up so far has nearly thousand kinds, and with hundreds of speed increment in every year.
Because people, mouse have on physiology than big-difference, mouse model also has its limitation.Carry out the gene knockout operation with large animal and can save the step of loaded down with trivial details separating animal's embryonic stem cell, and avoided mouse to produce chimeric trouble, can enhance productivity greatly.In addition, domestic animal such as pig, ox, sheep etc. are the more approaching mankind on dissection and physiology, do disease animal model more near the people with them.For example knock out the bladder fibrosis transmembrane and induce the mouse model of regulatory factor (Cfrt) gene to fail obviously to show pulmonary disorder feature under similar people's concrete conditions in the establishment of a specific crime, this mainly is to exist on lung's physiology of people and mouse to cause than big-difference.But the sheep that knocks out the Cfrt gene may produce the Fibrotic accurate model of similar human bladder, because lung's physiology of people and sheep is very similar.
The gene targeting research of large animal lags far behind mouse, mainly be because other animal can't be set up ES clone, and somatic cell clone technique is just carried out after clone sheep " Dolly " in 1997 birth.In June, 2000, first somatic cell gene target practice sheep that produces by nuclear transplantation is born in the world.The people such as McCreath of PPL company have reported that on Nature selection COL1A1 gene pairs sheep placenta inoblast carries out gene targeting, obtain three survival lamb (McCreath etc., Production of gene targeted sheep by nucleartransfection from cultured somatic cells.Nature, 2000,405:1066-1069).This result indicates fixed point change gene structure, rectification of defects gene, knocks out the beginning of genetic engineering methods such as deleterious gene.Utilize the transgenic sheep expression of exogenous gene amount of this method preparation can reach 60g/L milk, opened up new era that transgenic animal produce.In February, 2002, people such as Lai Liangxue have reported with clone's method at Science and have produced the clone pig that knocks out α-1.3-galactosidase gene that the existence of terminal α-1.3 galactoside (Gal) antigenic determinant on pig cell surface is the root that subacute rejection takes place when carrying out xenotransplantation with pig.
Summary of the invention
The preparation method that the purpose of this invention is to provide the transgenic cattle that a kind of milk composition is improved.
The preparation method of transgenic cattle provided by the present invention, be that the linearizing targeting vector that carries the beta-lactoglobulin gene that inserts sudden change is combined with somatocyte by homologous recombination, form the mediated cell of beta-lactoglobulin gene silencing, obtain reconstructed embryo in the enucleation oocyte with mediated cell nuclear importing ox, reconstructed embryo is moved in the replace-conceive cow uterus, the filial generation that obtains is transgenic cattle again.
The nucleotide sequence that the described targeting vector that carries the beta-lactoglobulin gene of sudden change can have sequence 1 in the sequence table; Reconstructed embryo in the described immigration replace-conceive cow uterus is embryo blastula stage.
Described somatocyte can be bovine fetal fibroblast; Described bovine fetal fibroblast can be ear inoblast, skin flbroblast, uterine tubal epithelium cell, granulosa cell or the adult inoblast etc. of ox fetus, is preferably the ear inoblast of ox fetus.
The linearizing targeting vector transfection bovine fetal fibroblast method that carries the beta-lactoglobulin gene of sudden change be can be conventional biology transfection method such as electrotransfection method, liposome transfection method, coprecipitation of calcium phosphate method, be preferably the electrotransfection method.
The condition of described electrotransfection is: DNA concentration is 18-22 μ g/mL, and strength of electric field is 1.2kV/cm, and the burst length is 7ms.
For improving transgene efficiency, described enucleation oocyte does not contain first polar body.
The method that the described mediated cell nuclear that will be integrated with the targeting vector of the linearizing beta-lactoglobulin gene that carries sudden change imports the enucleation oocyte of ox is a microinjection.
The present invention utilizes the method for homologous recombination and somatic cell clone that the linearizing somatic cell nuclear that carries the targeting vector of the beta-lactoglobulin gene that inserts sudden change is imported in the enucleation oocyte of ox, make it with cell chromosome in the homologous target sequence recombinate, finally obtain the filial generation of beta-lactoglobulin gene silencing.Milk with the transgenic cattle of method acquisition provided by the present invention is produced does not contain beta-lactoglobulin in its milk composition, is similar to people's milk more, and is both nutritious, can not cause the anaphylaxis to milk again, makes milk have edible widely crowd.The present invention has higher actual application value and vast market prospect.
Description of drawings
Fig. 1 is the production scheme of somatic cell clone beta-lactoglobulin gene knockout transgenic cattle
Fig. 2 makes up synoptic diagram for the beta-lactoglobulin gene knockout carrier
Fig. 3 is the PCR detected result of transgene clone ox
Fig. 4 is the PCR detected result of transgene clone ox
Fig. 5 is the Southern qualification result of transgene clone ox
Fig. 6 is the Southern qualification result of transgene clone ox
Embodiment
Be ordinary method among the following embodiment if no special instructions, agents useful for same and medicine are if no special instructions all available from Sigma company.
The production and the molecular Biological Detection of the transgenic cattle of embodiment 1, somatic cell clone beta-lactoglobulin gene knockout
The Production Flow Chart of somatic cell clone beta-lactoglobulin gene knockout transgenic cattle as shown in Figure 1, detailed process may further comprise the steps:
1, the structure of beta-lactoglobulin gene knockout targeting vector
Beta-lactoglobulin gene knockout carrier building process as shown in Figure 2, concrete grammar is as follows:
Genomic dna with bovine fetal fibroblast is a template, at primer pLeft upper:5 '-ca
Gcggccg cTagactgtgtgaatgcccactgac-3 ' (band underscore part base is a restriction enzyme NotI recognition site) and primer pLeft lower:5 '-ctc
TcgagcUnder the guiding of ttcatggtctgggtgacaatgag-3 ' (band underscore part base is a restriction enzyme XhoI recognition site), the dna fragmentation of pcr amplification 2488bp, the PCR reaction conditions is: 94 ℃ of 5min; 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 150s, 30 circulations; 72 ℃ of 7min.Reaction finishes the back PCR product is carried out the detection of 0.7% agarose gel electrophoresis, reclaim the specific PCR product of 2488bp, it is cut the back with restriction enzyme Not I with Xho I enzyme is connected with the carrier pLox that cuts through the same enzyme enzyme, to connect product Transformed E .coli DH5 α competent cell then, after blue hickie screening, select white single bacterium colony upgrading grain, and carry out the enzyme evaluation of cutting and check order, obtain containing the segmental recombinant vectors of purpose, called after pLox-BLG-Left has comprised the promoter region and first exon of beta-lactoglobulin gene in this plasmid.
Genomic dna with bovine fetal fibroblast is a template, under the guiding of primer pRight upper:5 '-tcattgtcacccagaccatgaag-3 ' and primer pRight lower:5 '-tgccaccgagcggctgtcctgg-3 ', the dna fragmentation of pcr amplification 6326bp, the PCR reaction conditions is: 94 ℃ of 5min; 94 ℃ of 30s, 66 ℃ of 30s, 72 ℃ of 7min, 30 circulations; 72 ℃ of 10min.Reaction finishes the back PCR product is carried out the detection of 0.7% agarose gel electrophoresis, reclaim the specific PCR product of 6326bp, itself and carrier pMD18-T (had the G418 resistant maker gene, TaKaRa company) connects, to connect product Transformed E .coli DH5 α competent cell then, after blue hickie screening, select white single bacterium colony upgrading grain, and carry out the enzyme evaluation of cutting and check order, obtain containing the segmental recombinant vectors of purpose, called after pMD18-Right has comprised part first exon and the coding region of beta-lactoglobulin gene in this plasmid.
PMD18-Right is cut the back with restriction enzyme Xba I with Sal I enzyme to be connected with the recombinant vectors pLox-BLG-Left that cuts through the same enzyme enzyme, to connect product Transformed E .coli DH5 α competent cell then, after blue hickie screening, select white single bacterium colony upgrading grain, and carry out the enzyme evaluation of cutting and check order, obtain beta-lactoglobulin gene knockout targeting vector, called after pLox-BLG-KO, it is carried out nucleotide sequencing, this carrier has the nucleotide sequence of sequence 1 in the sequence table, compare with the nucleotide sequence of normal beta-lactoglobulin gene, the open reading frame of beta-lactoglobulin gene has obtained to insert sudden change on the carrier.With plasmid vector pLox-BLG-KO behind restriction enzyme Not I linearization for enzyme restriction, carrying out 0.7% agarose gel electrophoresis detects, reclaim the also linear fragment of purifying 16874bp with QIAEX II test kit (QIAGEN company), it is dissolved in the ultrapure water of sterilization-20 ℃ of preservations.Holding 6-2487 position dna sequence dna from 5 ' in this sequence is to comprise the promoter region sequence of BLG gene and the homology left arm sequence of first exon, from the neomycin gene of 5 ' end 2562-4335 position dna sequence dna for inserting, from 5 ' end 4382-10702 position dna sequence dna is the homology right arm sequence that comprises BLG Gene Partial first exon and encoding sequence thereof, is the coding region of tk gene from 5 ' end 11254-12450 position dna sequence dna.
2, the foundation of ox fetus ear fibroblast
Select the holstein cow fetus (available from milk cow center, Beijing) at 5 monthly ages for use, take off the fetus ear, with PBS and 70% alcohol wash number all over after, shred into volume and be about 1mm
3Fritter, plant piece after using DMEM substratum (Invitrogen company) to clean 2 times again is 75cm in the volume that 3mL DMEM substratum (containing 10%FBS) is housed in batches
3Culturing bottle in, treat to add DMEM (containing 10%FBS) again to 10mL after tissue block adherent is firmly, be placed on 37 ℃, 5%CO
2Incubator in cultivate 6-7d, every 2d changes liquid 1 time, treat that cell growth converges after, with 0.25% trypsin Invitrogen company) digest and go down to posterity 2-3 time again, liquid nitrogen is preserved (the frozen storing liquid composition is: 60%DMEM, 30%FBS and 10%DMSO), obtains ox fetus ear fibroblast.
3, fibroblastic electrotransfection of ox fetus ear and screening
Collection is in the ox fetus ear inoblast of logarithmic phase, and HeBS (contains 140mmol/L NaCl, 5mmol/L KCl, 0.75mmol/L Na with electric shock liquid
2HPO
4, 6mmol/L glucose and 25mmol/L Hepes) and re-suspended cell to concentration is 5 * 10
6Individual/mL, in DNA concentration is 20 μ g/mL, strength of electric field is 1.2kV/cm, burst length is to shock by electricity under the condition of 7ms, linearizing fragment transfection ox fetus ear inoblast with carrier pLox-BLG-KO, adding 800 μ g/mL G418 and 2uM/L Gancvilor (Si gma) screen as screening pressure behind the 48h, every 2-3d changes liquid 1 time, behind the 14d with 0.25% trypsin trypsin) digestion collects clone's point cell and continues with 300 μ g/mLG418 and 2uM/L Gancvilor screening 4-5 generation, treating that growth converges also to be used for body-cell neucleus transplanting behind the serum starvation 2-4d.
4, the maturation of ovocyte is cultivated
With the ovary (taking from the slaughterhouse, surrounding area) of the ox that grows up with 37 ℃ PBS liquid cleaning three times after, with diameter is that to extract diameter be the ovarian follicle of 2-8mm for the syringe needle of 0.7mm, the recovery form is even, the ovarian cumulus-ovocyte-complex body (COCs) of compact structure, it (is added 10%FBS with ripe liquid on the basis of M199,0.01U/mL bFSH, 0.01U/mL bLH and 1 μ g/mL estradiol) wash twice, then ovarian cumulus-ovocyte-complex body is put into four orifice plates that contain ripe liquid by 50-60 piece/hole, place 38.5 ℃, 5%CO
2After maturation is cultivated 18-20h in the incubator, after sophisticated ovocyte put into the pipe vibration 2-3min that contains 0.1% Unidasa, blow and beat gently with Glass tubing again, cumulus cell and ovocyte are broken away from fully, select complete form, tenuigenin evenly and the ovocyte of discharging first polar body be cytosol receptor.
5, the vitro culture of nuclear transplantation and clone embryos
The ovocyte that has first polar body that step 4 is obtained moves in the operation liquid (having added 10%FBS and 7.5 μ g/mL cytochalasin Bs on the basis of M199), under 200 power microscopes, above polar body, zona pellucida cut an osculum with a glass needle, be that the Glass tubing of 20 μ m is absorbed the karyomit(e) in the ovocyte of first polar body and below thereof in the lump with internal diameter again, again with the M199 liquid nutrient medium that contains 20%FBS give a baby a bath on the third day after its birth all over after, place 38.5 ℃, 5%C0
2Standby in the incubator.The transgenic cell of the serum starvation 2-4d that step 3 is obtained is with 0.25% trypsin trypsin) digest 2-4min, selecting diameter is the fetus ear inoblast nuclear of 10-12 μ m, with 20 μ m diameter Glass tubings it is moved in the non-nucleus egg mother cell zona pellucida, put it into then and contain 0.3M N.F,USP MANNITOL (Mannitol), 0.15mmol/L Ca
2+With 0.15mmol/L Mg
2+Solution in, put into integration slot behind the 3-5min, rotating ovum makes donorcells nuclear vertical with electric field with the ovocyte contact surface, be 2.5kV/cm, burst length to be that 10 μ s, pulse number are after 2 times, recurrent interval are to merge (fusion instrument is the ECM-2001 of BTX company) under the condition of 1s simultaneously in the field intensity of DC pulse, rapidly reconstructed embryo is moved into and contain in the M199 liquid nutrient medium of 10%FBS, observe fusion rate after placing 0.5h, select the fusion embryo and carry out next step activation processing.Reconstructed embryo is put into 5 μ mol/L ionomycins, and (Ionomycin Sigma) in the liquid, moves to behind the 4min in the 1.9mmol/L6-DMAP liquid, moves into behind the 4h to contain in the CRlaa liquid of 5%FBS, at 38.5 ℃, 5%CO again
2Cultivate in the incubator, behind 2d and 7d, observe embryo's development condition respectively.
6, embryo transfer and gestation detect
Clone's blastaea of the 7d that form is good moves in the horn of uterus of the recipient cattle of the same period.30d after transplanting carries out B ultrasonic to receptor cow and detects determining the situation of being impregnated, and the 60d after transplanting and 90d carry out rectum and detect to determine pregnancy rate respectively.15 of co-transplantation recipient cattle, the rectum of transplanting back 60d detects and shows wherein 8 pregnancies.2 transgene clone ox births are finally arranged, and calving rate is 13.3%.
7, the molecular biology identification of transgene clone ox
1) PCR identifies
The transgenosis that acquisition step 6 obtains and the tissue sample of non-transgenic clened cows ear, extract genomic dna according to a conventional method, and be template with it, respectively at primer to 1 (primer T1upper:5 '-GTCTGAAGGCAGCTCGCTGTG-3 ' and primer T1lower:5 '-GGGAAACCAGGGCACGCAAAG-3 ') and primer under the guiding to 2 (primer T2upper:5 '-TATTGGCAGGCAGATTCTTTACCAC-3 ' and primer T2 lower:5 '-tgttaagcgggtcgctgca-3 '), genomic dna to transgenosis and non-transgenic clened cows carries out the PCR detection, with the genomic dna of common holstein cow as negative control, with the knockout carrier pLox-BLG-KO of beta-lactoglobulin gene as positive control.Primer to 1 PCR reaction conditions is: 94 ℃ of 5min of elder generation; 94 ℃ of 30sec then, 61 ℃ of 30sec, 72 ℃ of 180sec, totally 30 circulations; Last 72 ℃ of 7min.Reaction finishes the back PCR product is carried out the detection of 0.7% agarose gel electrophoresis, (swimming lane 1 is 1kb Marker for detected result such as Fig. 3, swimming lane 2 positive plasmid pLox-BLG-KO, swimming lane 3 is a sterile purified water, swimming lane 4 is common holstein cow, swimming lane 5 and swimming lane 6 are beta-lactoglobulin gene knockout clened cows) shown in, show that the transgene clone ox can amplify the wild-type band of 1143bp and the insert type band of 3087bp, and common holstein cow only can expand the wild-type band that 1143bp.Primer to 2 PCR reaction conditions is: 94 ℃ of 5min of elder generation; 94 ℃ of 30sec then, 61 ℃ of 60sec, 72 ℃ of 30sec, totally 30 circulations; Last 72 ℃ of 7min.Reaction finishes the back PCR product is carried out the detection of 0.7% agarose gel electrophoresis, (swimming lane 1 is 1kb Marker for detected result such as Fig. 4, swimming lane 2 positive plasmid pLox-BLG-KO, swimming lane 3 is a sterile purified water, swimming lane 4 is common holstein cow, swimming lane 5 and swimming lane 6 are beta-lactoglobulin gene knockout clened cows) shown in, show that transgene clone ox and normal holstein cow all can not amplify the tk gene band of 1182bp, obtain and can only increase on carrier.
2) Southern identifies
Get the genomic dna of about 10 μ g transgene clone the ears of an ox or cow portions tissue, cut digestion with restriction enzyme Eco72I enzyme, the 30V low voltage electrophoresis, after changeing film, carry out Southern hybridization, hybridizing used probe is that α-P32dCTP is isotope-labeled, pLox-BLG-KO carrier recovery product through restriction enzyme XhoI and XbaI enzyme cutting digestion, with the genomic dna of common holstein cow as negative control, with knockout carrier pLox-BLG-KO as positive control, the hybridization positive signal is the 3.2kb fragment, the result is (swimming lane 1 as shown in Figure 5,2 is two beta-lactoglobulin gene knockout clened cows, swimming lane 3 is common holstein cow, swimming lane 4 is 1 copy positive plasmid, swimming lane 5 is 5 copy positive plasmids, swimming lane 6 is 10 copy positive plasmids) genomic dna of about again 10 μ g transgene clone the ears of an ox or cow portions tissue, cut digestion with restriction enzyme MscI enzyme, the 30V low voltage electrophoresis, after changeing film, carry out Southern hybridization, hybridizing used probe is that α-P32dCTP is isotope-labeled, cut the pLox-BLG-KO carrier recovery product of digestion through restriction enzyme SalI and HindIII enzyme, with the genomic dna of common holstein cow as negative control, with knockout carrier pLox-BLG-KO as positive control, the hybridization positive signal is the 5.6kb fragment, (swimming lane 1 as shown in Figure 6,2 is two beta-lactoglobulin gene knockout clened cows, and swimming lane 3 is common holstein cow, and swimming lane 4 is 1 copy positive plasmid, swimming lane 5 is 5 copy positive plasmids, and swimming lane 6 is 10 copy positive plasmids).The above results shows at this two beta-lactoglobulin and knocks out the homologous recombination that expection has taken place on the individual genomic BLG locus.Southern Blot qualification result and PCR detected result are in full accord.
Further tracking test shows, after the transgenic cattle that obtains with method of the present invention grows up to, do not contain beta-lactoglobulin in its excretory milk, but other composition and ordinary milk does not have difference.
Sequence table
<160>1
<210>1
<211>16874
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
Claims (8)
1, a kind of preparation method of transgenic cattle, be that the linearizing targeting vector that carry the beta-lactoglobulin gene of sudden change of base sequence shown in sequence in the sequence table 1 combined with bovine fetal fibroblast by homologous recombination, form the mediated cell of beta-lactoglobulin gene silencing, obtain reconstructed embryo in the enucleation oocyte with mediated cell nuclear importing ox, with in the described reconstructed embryo immigration replace-conceive blastula stage cattle uterus, the filial generation that obtains is transgenic cattle again.
2, the preparation method of transgenic cattle according to claim 1 is characterized in that: described bovine fetal fibroblast is ear inoblast, skin flbroblast, uterine tubal epithelium cell or cumulus cell.
3, the preparation method of transgenic cattle according to claim 2 is characterized in that: described bovine fetal fibroblast is the ear inoblast.
4, according to the preparation method of claim 1 or 2 or 3 described transgenic cattles, it is characterized in that: described is electrotransfection method, liposome transfection method or calcium phosphate precipitation method with the linearizing targeting vector transfection bovine fetal fibroblast method that carries the beta-lactoglobulin gene of sudden change.
5, the preparation method of transgenic cattle according to claim 4 is characterized in that: described is the electrotransfection method with the linearizing targeting vector transfection bovine fetal fibroblast method that carries the beta-lactoglobulin gene of sudden change.
6, the preparation method of transgenic cattle according to claim 5 is characterized in that: the condition of described electrotransfection is: DNA concentration 18-22 μ g/mL, strength of electric field 1.2kV/cm, burst length 7ms.
7, according to the preparation method of claim 1 or 2 or 3 described transgenic cattles, it is characterized in that: described enucleation oocyte has first polar body.
8, according to the preparation method of claim 1 or 2 or 3 described transgenic cattles, it is characterized in that: the method that the described mediated cell nuclear that will be integrated with the targeting vector of the linearizing beta-lactoglobulin gene that carries sudden change imports the enucleation oocyte of ox is a microinjection.
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| CN102321609B (en) * | 2010-09-09 | 2014-04-16 | 山东省农业科学院奶牛研究中心 | Method for obtaining foot-and-mouth disease (FMD) resistant transgenic cattle by knocking out foot-and-mouth disease virus (FMDV) receptor integrin beta6 subunit gene |
| CN102352379B (en) * | 2010-10-20 | 2013-11-13 | 山东省农业科学院奶牛研究中心 | Method for obtaining foot-and-mouth-disease resisting transgenic cow trough knocking out foot-and-mouth-disease virus receptor integrin alpha v subunit gen |
| CN101979607A (en) * | 2010-11-08 | 2011-02-23 | 山东省农业科学院奶牛研究中心 | Method for preparing foot-and-mouth disease virus-resistant RNAi transgenic livestock |
| CN102212545B (en) * | 2011-04-07 | 2013-05-08 | 北京济福霖生物技术有限公司 | Method for knocking out cattle beta-lactoglobulin gene by using zinc finger nucleases (ZFNs) |
| CN109321600A (en) * | 2018-10-19 | 2019-02-12 | 中国农业大学 | A kind of method and its application of ox that cultivating production low irritability milk |
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