CN1745626B - Modified cell line of male mammal, its production and use thereof - Google Patents
Modified cell line of male mammal, its production and use thereof Download PDFInfo
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Abstract
A modified somatic cell line of male mammal features that the marker protein gene chosen from fluorescin gene, beta-galactosidase gene, leuciferinase gene and beta-glucuronidase gene is integrated in its chromosome Y. Its preparing process and its application in culturing male mammal are also disclosed.
Description
Technical field
The present invention relates to a kind of modified cell line of male mammal and preparation method thereof and application, particularly a kind of modified cell line of male mammal and preparation method thereof and the method for utilizing this somatocyte system to cultivate boar.
Background technology
The animal transgenic research is the research focus of biological technical field, and its range of application has been penetrated into numerous areas such as fundamental research, agricultural, medicine.The animal transgenic research is meant by various laboratory facilities, and the foreign gene structure that makes up according to specific purpose is incorporated in the genome of host animal, expresses in the growth course of animal, and passes to offspring's process by sexual cell.This animal that stably is integrated with the foreign gene of artificial importing in genome claims transgenic animal.
In the reproductive process of domestic animal, there are two kinds of sperms that contain X or Y chromosome in the seminal fluid of male domestic animal, the sperm that contains X chromosome in fertilization process combines the offspring that produced for female with ovum, and the sperm that contains Y chromosome combine the offspring who is produced with ovum just be male.And in fertilization process sperm and ovum in conjunction with being at random, so be at random and can not prediction in the sex of the normal reproductive process generation of neutrons of domestic animal, and in the domestic animal because of the difference of sex, the economic benefit difference that it brought is huge.People thirst for accessing female individuals as much as possible in the reproductive process of domestic animal, wish to obtain hen as much as possible as people in the breeding of chicken, and people always wish to obtain cow etc. in the reproductive process of milk cow.With the milk cow is example, the cow of new birth and the price of bull differ more than ten times, because the huge spread of economic interests, people have invented the various methods of selecting or judge the domestic animal sex in advance, for example utilize cell separation technology that the sperm that contains Y chromosome in the livestock semen is separated, be fertilized with the seminal fluid after separating, thereby improve the ratio of female domestic animal among the offspring, but this method needs the very expensive separating device of price, and isolating speed is also very slow, can not reach the purpose of scale operation.Motility of sperm after the separation also obviously descends, and the sperm after separating can not reach 100% separating effect, is fertilized with it and can only improves the ratio of female offspring to a certain extent, and is not only time-consuming, and cost is very high.People also attempt will containing with electrophoresis, method such as centrifugal the sperm of X chromosome and separate with the sperm that contains Y chromosome, but up to the present, also can effectively make the complete isolating technology of X.Y sperm without any a kind of method.People also attempt getting some tissues and carry out pcr analysis and judge its sex from livestock embryo, but this method has very big damage to the embryo, and this analysis needs the long period, and the embryo is subjected to strict restriction the immigration acceptor time in external time and embryo, if in time can not be synchronous, transplanting efficient be affected.In order to address this problem, the have to temporary transient freezing preservation of embryo that will carry out pcr analysis of people is treated to thaw after PCR result comes out again, and has increased the difficulty of operation so greatly, and the embryo is produced further injury.
1997, the report that the first somatic cell clone sheep " Dolly " is born caused the strong interest of various circles of society, also indicated the successful foundation of a kind of new technology-mammal body-cell neucleus transplanting technology of biological field simultaneously.The first of the same year report utilizes the birth of the Transgenic Sheep " Polly " that somatic cell nuclear transfer technique cultivates to be only the real milestone in animal transgenic research field, and it has raised the new page that somatic cell nuclear transfer technique is used for the big domestic animal of production transgenosis.In the period of subsequently several, the transgenic cattle, goat and even the site-directed integration that utilize somatic cell nuclear transfer technique to cultivate have the sheep of foreign gene to be born in succession.Over nearly 2 years, scientist cultivates the clone pig that fixed point knocks out single copy (singly knocking out) and two copy (two knocking out) α-1,3 galactoside transferase genes again in succession, and this The Application of Technology field is constantly expanded.
Somatic cell nuclear transfer technique and somatic cell gene transfer techniques are integrated as the production transgenic animal and have opened up an effective way.The maximum characteristics of this technological line are mainly reflected in the transgenosis step are advanceed to the somatic cell culture stage.Cytogene shifts and is meant by electroporation liposome, transfection methods such as calcium phosphate precipitation and microinjection, the external source target gene is integrated in the cellular genome, and utilize specific selection markers, and a large amount of amplifications, enrichment transgenic cell, thus transgenosis cell strain obtained.The cloned animal that directly utilizes this fixed somatocyte that is integrated with goal gene to cultivate for nuclear donor must all be transgenic animal.The advantage of producing breeding transgenic livestock by somatic cell nuclear transfer technique is very obvious: at first, this method is drawn materials easy, and ovocyte can obtain from the slaughterhouse, and the animal somatic cell source is abundant especially.Secondly and since the institute animal that produces most be transgenic animal, thereby compressed the quantity of replace-conceive dam significantly, reduced production cost.Can shorten acquiring the time that forms transgenic animal group system of former generation transgenic animal with throughput; More valuable is to utilize this technology can also obtain the big domestic animal of gene site-directed modification.Under precondition, this is that other transgenic method is beyond one's reach.
(florescence in-situ hybridization FISH) is a kind of on-radiation in-situ hybridization method to fluorescence in situ hybridization.With special fluorescein-labelled nucleic acid (DNA) probe, can on karyomit(e), cell and tissue slice sample, carry out DNA hybridization, whether exist the most effective to the particular sequence that detects DNA in the cell or RNA.The fluorescein-labelled of probe can be adopted directly and the method for indirect labelling.Indirect labelling is to adopt biotin labeled dUTP (biotin-dUTP) to carry out mark through nick-translation, the antibody that is associated with the antibiotin of fluorescein with lotus root after the hybridization detects, can also utilize simultaneously several processing of taking turns avidin-fluorescein, biotinylated resisting-avidin, avidin-fluorescein, fluorescent signal is amplified, thereby can detect the fragment of 500bp.And direct labelling method is direct and probe nucleotide or phosphopentose skeleton covalent attachment with fluorescein, or when the nick-translation label probe fluorescein ribonucleoside triphosphote is mixed.Direct labelling method step when detecting is simple, but owing to can not carry out the signal amplification, so sensitivity is not as the method for indirect labelling.
The innovation and creation content
The purpose of this invention is to provide a kind of modified cell line of male mammal.
Modified cell line of male mammal provided by the present invention, its Y chromosome is integrated with the labelled protein gene.
Described labelled protein gene is preferably the fluorescence protein gene that strengthens green fluorescence protein gene, green fluorescence protein gene, beta-galactosidase gene, luciferase gene, GRD beta-glucuronidase gene or other color.
For the ease of the screening of clone, described Y chromosome also is integrated with selectable marker gene; The preferred neomycin resistance gene of described selectable marker gene.
Second purpose of the present invention provides a kind of method for preparing above-mentioned modified cell line of male mammal.
The method of the above-mentioned modified cell line of male mammal of preparation provided by the present invention may further comprise the steps:
1) will contain in the somatocyte of labelled protein expression carrier importing boar;
2) distinguished sequence with described boar Y chromosome is a probe, carries out fluorescence in situ hybridization with somatocyte in the step 1), and screening obtains having the boar somatocyte of the Y chromosome of being modified by described labelled protein.
The described labelled protein expression carrier that contains can be passed through electroporation, liposome, and transfection methods such as calcium phosphate precipitation and microinjection import in the somatocyte of boar.
Described labelled protein gene can be selected from fluorescence protein gene, beta-galactosidase gene, luciferase gene or GRD beta-glucuronidase gene, wherein fluorescence protein gene is preferably and strengthens green fluorescence protein gene or green fluorescence protein gene, can certainly be the fluorescence protein gene of other color.
For the ease of screening, the expression vector in the step 1) also contains selectable marker gene; Described selectable marker gene is preferably neomycin resistance gene.
In order to guarantee that described labelled protein gene and described selectable marker gene express simultaneously, described expression vector also contains IRES (internal ribosome entry site of picornavirus) sequence.
The probe that carries out fluorescence in situ hybridization with somatocyte in the described step 1) also has labelled protein gene fragment and/or selectable marker gene fragment.
Described boar can be Mammalss such as male domestic animal or mouse; Described male domestic animal can be bull, ram, buck or boar etc.
When described boar was bull, the distinguished sequence of described Y chromosome was any distinguished sequence of the Y chromosome of bull; When described boar was ram, the distinguished sequence of described Y chromosome was any distinguished sequence of the Y chromosome of ram; When described boar was buck, the distinguished sequence of described Y chromosome was any distinguished sequence of the Y chromosome of buck; When described boar was boar, the distinguished sequence of described Y chromosome was any distinguished sequence of the Y chromosome of boar; When described boar is male mouse, the distinguished sequence of the Y chromosome of described male mouse be male mouse any distinguished sequence of Y chromosome.
Above-mentioned expression vector all can make up according to ordinary method.
In the described fluorescence in situ hybridization, the fluorescein-labelled of probe can be adopted direct labelling method or indirect labelling method.
The 3rd purpose of the present invention provides a kind of method of cultivating boar.
The method of cultivation provided by the present invention boar, be with above-mentioned modified boar somatocyte as for the nucleome cell, obtain boar through the somatic cell clone method.
Described boar can be Mammalss such as male domestic animal or mouse; Described male domestic animal can be bull, ram, buck or boar etc.
Described somatic cell clone method is conventional mammalian somatic cell cloning process.
Principle of the present invention is not that the X.Y sperm is separated, but make Y chromosome expression specific mark albumen (as fluorescin) among the after fertilization embryo by genetic marker to Y chromosome, distinguish and to have or not the expression of labelled protein (fluorescin) among the embryo and then embryo's sex is distinguished, the proteic embryo of presentation markup is a male embryo, and the proteic embryo of presentation markup is not a female embryo.
Utilize clone of the present invention to obtain boar through the somatic cell clone method, with the embryo of this boar after fertilization with presentation markup albumen, can reduce the cost of screening Mammals descendant sex greatly, and make the rate of accuracy reached 100% of screening, and the embryo is not had any damage, will improve mammiferous breeding efficiency greatly.
Description of drawings
Fig. 1 is the transfection fragment of double alternative carrier pCE-EGFP-IRES-NEO
Embodiment
Embodiment 1, Y chromosome are modified with the somatic preparation of the breeding oxen that strengthens green fluorescence protein gene and neomycin resistance gene
One, the foundation of breeding oxen clone
Get the ear skin tissue of holstein cow breeding oxen, clean behind ear's lower edge dorsal part unhairing with 70% alcohol wash, pick from ear's lower edge dorsal part with blade again that to get area be 1cm
2About skin, place 0 ℃ DMEM/F12 to transport the laboratory as early as possible back, with PBS and 70% alcohol wash number all over after shred into 1mm
3About fritter, DMEM/F12 plants piece in the 25cm that contains 1mL DMEM/F12+10%FBS after cleaning 2 times in batches
2Culturing bottle in, treat to add DMEM/F12+10%FBS to 6mL again after tissue block adherent is firmly, in 37 ℃, 5%CO
2Incubator is cultivated 6-7d, and every 2d changes liquid 1 time, treat that the cell growth converges after, with 0.25% trypsin trypsin) had digestive transfer culture 2-3 time, frozen with DMEM/F12+20%FBS+10%DMSO in batches.Like this, through former foster, the cultivation of going down to posterity of being commissioned to train, vitro culture operation such as freezing, set up the breeding oxen fibroblast.
Two, make up double alternative carrier pCE-EGFP-IRES-NEO
1, the structure of double alternative carrier
With EcoRI, XbaI double digestion plasmid pCE321-FL (available from Clontech company) and plasmid pCMV-EGFP-IRES-NEO (available from Clontech company), after the neomycin resistance gene (Neor) that enhancing green fluorescence protein gene (EGFP) after cytomegalovirus (CMV) promotor among the plasmid pCMV-EGFP-IRES-NEO and internal ribosome entry site (IRES) are carried is inserted into 5 ' the control region pCE321 that is made up of cytomegalovirus enhanser (CMV-IE Enhancer) and people's EF-1 α promotor (pEF321) among the plasmid pCE321-FL, substitute the CMV promotor with pCE321, form double alternative carrier pCE-EGFP-IRES-NEO.Cut evaluation through enzyme, show that the double alternative carrier pCE-EGFP-IRES-NEO and the test design that build meet fully.
Wherein, neomycin resistance gene is used for the screening and the enrichment of transgenic cell; The enhancing green fluorescence protein gene is used for filtering out effectively the transgenosis blastaea and is used for embryo transfer, with the cloned animal of guaranteeing to cultivate is transgenic animal entirely, and IRES (internal ribosome entry site of picornavirus) guarantees that EGFP and Neor can express simultaneously.
Plasmid pCE-EGFP-IRES-NEO reclaims big fragment through the QIAEX of QIAGEN company II test kit purifying behind SalI and AscI double digestion, obtain linear double alternative carrier, is dissolved in the sterilization ultrapure water, and-20 ℃ of preservations are used for transfectional cell.
Three, the screening of gene transfection and transfectional cell
In available following two kinds of methods any one carries out gene transfection.
1, the cytogene transfer method of electroporation mediation
(1) 2d before transfection is with trypsin solution digestion breeding oxen fibroblast.With 1 * 10
6Individual cell is transferred in the 100ml culturing bottle, adds the DMEM nutrient solution that 4ml contains 10% calf serum.Put 37 ℃, 5%CO
2Cultivate in the incubator.
(2) before the transfection, under inverted microscope, observe cultured cells, select growth cell vigorous, that form is good and be used for transfection.
(3) use the trypsin solution peptic cell.With the centrifugal 5min sedimentation cell of 400g.PBS with pH7.4 washes 1 time with cell precipitation.Cell is suspended among the 1mlPBS again.
(4) containing 5 * 10
5The transfection plasmid DNA that adds the 1-10ug purifying in the cell suspension of cell.Change over to after the mixing in the electric shock cup.
(5) with the strength of electric field of 1.2KV/cm and the burst length of 1ms, the electric shock cell.
(6) cell after will shocking by electricity changes in the culturing bottle of 100ml.Adding 4ml contains the DMEM nutrient solution of 10% calf serum.In CO
2After cultivating 1-2d in the incubator, cell growth state is recovered, and it is 200-800ug/ml that adding G418 makes its final concentration.In 37 ℃, 5%CO
2After continuing in the incubator to cultivate 6-14d, select the positive colony cell.
2, lipid mediated gene transfer
(1)-(3) identical with step 1.
(4) in two 1.5ml centrifuge tubes, prepare following solution respectively:
A: the 1-5ug transfection is dissolved in the DMEM nutrient solution that 100ul do not add serum with plasmid DNA.
B: the 20ul liposome is mixed in the DMEM nutrient solution that 80ul does not add serum.
(5) merge solution A and B, mix the back gently and under room temperature, place 20min.Then, add the nutrient solution that 800ul does not add serum.
(6) plasmid DNA, liposome and the mixture that do not add the nutrient solution of serum are dripped in culturing bottle, treat the cells transfected surface, in CO
2Cultivate 24h in the incubator.
(7) outwell transfection liquid, add 4ml and contain 10% DMEM complete culture solution.Continue to cultivate 36-48 hour in incubator, add G418 solution in culturing bottle, making its final concentration is 200-800ug/ml.In 37 ℃, 5%CO
2Continue in the incubator to cultivate.
(8) select the positive colony cell strain behind the 6-14d, forward to after the digestion in 96 orifice plates respectively and cultivate, treat that cell covers with after, each cell strain is got a part of cell cryopreservation, another part cell cultures is carried out the cell chromosome preparation.
Four, prepare the Metaphase Chromosome cell from the cell of adherent growth
Be approximately 70% and be in the cell of active condition in order to obtain degree of converging,, attached cell cultivated at preceding 1-2 days of sample preparations.The steps include:
1. check the situation that exists of mitotic cell.
A. adding colchicine acid amides to final concentration in culture is 0.1ug/ml, hatches 60-90 minute for 37 ℃.
B. digest with 0.2% trypsinase pair cell.Draw the mitotic cell of the change circle on the culturing bottle with sharp keen suction nozzle.
C. continue the Metaphase Chromosome cell is carried out hypotonic processing, be convenient to the preparation of karyomit(e) cell smear.
2.800g centrifugal 5-10 minute, sedimentation cell kept the 0.2-0.6ml supernatant, discards all the other supernatants.
3. cell is resuspended in the DMEM nutrient solution supernatant, carefully adds the 0.075mol/L KCL of 37 ℃ of preheatings of about 1/2 volume, the limit edged carefully stirs mixing.
4.37 ℃ hatched 12-20 minute.
5. 2 centrifugation cells set by step suspend cell precipitation again.
6. (formaldehyde: Glacial acetic acid: water=4: 1: 5) 10ml is with fixed cell, and the added-time will be stirred mixing dropwise to add freshly prepd ice-cold fixing agent.
7. cell was placed 5 minutes on ice.
8. 5 centrifugation cells set by step 6 repeat fixed cells set by step, and cell was placed 30 minutes on ice.
9. 5 and 6 repeat to fix 5 times set by step, do not need ice bath during fixing.
10. cell suspension is dropped on the washed in advance slide glass.
11.70%, 90% and 100% serial dehydration of alcohol, each 5 minutes, dry air then.
Five, the chromosomal in situ hybridization of transgenic cell (FISH):
Respectively with the 5.8Kb nucleotide sequence segment of bull Y chromosome specific gene Sry (testicular determining gene) (available from French National Agricultural Science research institute (INRI), gene numbering INRIBac8784) and the 10.2kb nucleotide sequence segments of the two mark of pCE-EGFP-IRES-NEO gene (behind NotI digested plasmid pCE-EGFP-IRES-NEO, the fragment that obtains through the recovery of the QIAEX of QIAGEN company II test kit purifying) for probe transgenic cell karyomit(e) is carried out in situ hybridization (FISH). concrete grammar is:
1, dna probe preparation
Utilize BioNick Labeling system test kit (Invitrogen) to be prepared as follows dna probe:
1) centrifuge tube is placed on ice, adds following component:
5ul 10 * dNTP mixture
The DNA of total amount 1ug
Add ddH
2O to 45ul
5ul 10 * enzyme mixture;
2) mix 12000rpm centrifugal 5 seconds;
3) 16 ℃ of water-baths, electrophoresis detection (the testing process middle probe places on ice, supspends), reach the institute clip size of wanting (360bp) after, add the 5ul stop buffer, termination reaction;
4) add 1/10 volume 3M NaAc and 2 times of volume precooling dehydrated alcohols, put into-70 ℃ of refrigerators after mixing 15 minutes.Centrifugal 10 minutes of 12000rpm, the dry air precipitation is used 50ul ddH
2O is resuspended, is stored in-20 ℃.
2, probe and sample sex change
1) probe sex change
Add in the probe 10ul hybridization solution (50% methane amide, 10% T 500,2 * SSC), piping and druming evenly, and is centrifugal, 65 ℃ of preheatings 5 minutes, in 75 ℃ of sex change 5 minutes, ice was put 5-10 minute (putting to hybridization) immediately.
2) slide sample sex change
The hybridization sample was in 50 ℃ of roasting sheets at least 2 hours.Sample places 70-75 ℃, among 70% methane amide/2 * SSC sex change 2-3 minute.In order sample was dewatered each 5 minutes through 70%, 90% and 100% ice ethanol series immediately.Drying at room temperature.
3, in situ hybridization
1) hybridization
The dna probe of sex change is dripped on the slide sample of sex change and dehydration, covered, the RubberCement mounting places 37 ℃ of hybridization of moist magazine spend the night (about 15-17h).
2) wash-out
1. hybridize next day, sample is taken out from 37 ℃ of incubators, remove cover glass.
2. the slide sample that will hybridize is in 42-50 ℃, and washing is 4 times among 50% methane amide/2 * SSC, each 5 minutes.
3. in 42-50 ℃, washing is 3 times among 1 * SSC, each 5 minutes.
4. at room temperature, with slide sample fine laundering in 2 * SSC.
3) immunofluorescence detects with signal and amplifies, and the steps include:
1. add 100-200ul confining liquid I (3%BSA/4 * SSC/0.1%Tween20) on sample, 37 ℃ incubation 15-30 minute.Add 100-200ul avidin-FITC again on sample, 37 ℃ incubation 30-60 minute.
2. take out sample, in 42-50 ℃, washing is 3 times among 4 * SSC/0.1%Tween20, each 5 minutes.
3. add 100-200ul confining liquid II:(3%BSA/4 * SSC/5% sheep blood serum/0.1%Tween20) on sample, 37 ℃ incubation 15-30 minute.Add 100-200ul antiavidin on sample, 37 ℃ incubation 30-60 minute.
4. 2.-1.-2. step of repeating step.
5. room temperature fine laundering in 2 * SSC.
6. slide takes out in washings, seasoning, and PI/antifade redyes, covered, fluorescence is observed down.Select and on same karyomit(e), hybridize the cells that bull Y chromosome distinguished sequence and 2 kinds of fluorescence probe signals of two mark genes are arranged, and to criticize the cell of freezing preservation together as somatic cell clone and nuclear donor cell.
Embodiment 2, the somatocyte that utilizes Y chromosome to be modified with the breeding oxen that strengthens green fluorescence protein gene and neomycin resistance gene are cultivated breeding oxen
1, the maturation of ovocyte is cultivated
Collect the ovary of the ox that grows up from the slaughterhouse, place 30 ℃ physiological saline in 4h, to deliver to the laboratory, after cleaning three times in 37 ℃ the PBS liquid, is the ovarian follicle of 2-8mm with the diameter for the 0.7mm syringe needle extracts diameter, the recovery form is even, the ovarian cumulus-ovocyte-complex body (COCs) of compact structure, with twice of ripe liquid (M199+10%FBS+0.01U/mLbFSH+0.01U/mL bLH+1 μ g/mL estradiol) washing, then ovarian cumulus-ovocyte-complex body is put into four orifice plates that contain ripe liquid with 50-60 piece/hole, at 38.5 ℃, 5%CO
2After maturation is cultivated 18-20h in the incubator, after sophisticated ovocyte being put into the pipe vibration 2-3min of 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.
2, the vitro culture of nuclear transfer procedure and clone embryos
The ovocyte that will have first polar body moves into and contains in the operation liquid of M199+10%FBS+7.5 μ g/mL cytochalasin B, under 200 power microscopes, above polar body, zona pellucida cut an osculum with glass needle, again with internal diameter be 20 μ m Glass tubing with first polar body with and the ovocyte of below in karyomit(e) absorb in the lump, the solution of putting into M199+20%FBS again give a baby a bath on the third day after its birth all over after, place incubator standby.
Serum starvation 2-4d passed through transgenic cell that Chromosomal in situ hybridization (FISH) identifies with 0.25% trypsin trypsin) digest 2-4min, is that the bull transgenic cell of 10-12 μ m moves in the ovocyte zona pellucida removed nuclear with 20 μ m diameter Glass tubings with diameter, putting it into balance in the Zimmerman liquid (matching while using) then puts into integration slot and rotates ovum and donorcells is contacted and vertical with electric field with ovocyte after 3-5 minute, be in the DC pulse field of 2.5kV/cm in field intensity simultaneously, in the burst length is 10 μ s, pulse number is 2 times, recurrent interval is after merging (fusion instrument is the ECM-2001 of BTX company) under the condition of 1s, after cultivating a few hours in the rapid M199+10%FBS of the immigration liquid, select the fusion embryo and carry out next step activation processing.Reconstituted embryo is put into 5mMA23178 (sigma C-7522) liquid change to the activation liquid 5 hours that contains 5ug/ml cytochalasin B+10ug/ml cycloheximide after 5 minutes, treat to change to after reconstituted embryo is activated CR1aa+5%FBS at 38.5 ℃, 5%CO
2Cultivate the blastocyst rate of observing the nuclear transplantation embryo after 7 days in the incubator in the nutrient solution, the result shows that clone's blastocyst rate is 20%-60%.
3, embryo transfer and gestation detect
The double-tagging gene clone blastaea of the 7d that form is good moves in the horn of uterus of 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, and pregnancy rate is 40%.
4, to cow in calf routinely method for breeding raise, through 280 days, cow in calf normal labor obtained the somatic cell clone breeding oxen.
Embodiment 3, Y chromosome are modified with the biological assay of the breeding oxen that strengthens green fluorescence protein gene and neomycin resistance gene
Gather transgenosis and non-transgenic clened cows ear and organize sample, extract genomic dna according to a conventional method.Carrying out PCR according to following method identifies and sex identification
1, PCR identifies
With one couple of PCR primers (upstream: 5 '-TGC AGT GCT TCA GCC GCT AC-3 ' downstream: 5 '-CTCAGG TAG TGG TTG TCG GG-3 ') transgenosis and non-transgenic clened cows genome DNA sample are carried out pcr amplification; with common holstein cow genomic dna as negative control, with double-tagging vector plasmid pCE-EGFP-IRES-NEO as positive control.The PCR response procedures is 94 ℃ of 5min; 94 ℃ of 30sec, 62 ℃ of 30sec, 72 ℃ of 30sec, 30 circulations; 72 ℃ of 7min.The result shows that the amplified production of transgenic cattle is the dna fragmentation of the about 384bp of total length, but not does not have this band in the amplified production of transgenic cattle.
2, marker gene is modified the expression of Y chromosome transgenic cattle and the evaluation of sex of children;
(1) the transgenosis bull that its Y chromosome that cultivation is obtained is modified by fluorescence protein gene and the cow of estrus synchronization carry out mating after four to seven days reclaims the intravital embryo of cow, places under the fluorescent microscope and observes, and has the embryo of fluorescence to choose with sending out.
With the PCR method its result is verified again, with one couple of PCR primers (upstream: 5 '-TGC AGT GCT TCAGCC GCT AC-3 ', downstream: 5 '-CTC AGG TAG TGG TTG TCG GG-3 ') this embryonic gene group DNA is carried out pcr amplification.Wherein, the PCR response procedures is: 94 ℃ of 5min; 94 ℃ of 30sec, 62 ℃ of 30sec, 72 ℃ of 30sec, 30 circulations; 72 ℃ of 7min.The result shows that this embryo's amplified production total length is about 384bp, illustrates that this embryo is a male embryo.
(2) get the seminal fluid that Y chromosome changes the transgenosis bull that fluorescence protein gene is arranged, join and contain heparin (50ug/ml), caffeine (0.01nmol/ml), dilution is washed twice (with 1800 rev/mins in the BO liquid of BSA (3.0mg/ml), centrifugal 8 minutes), after removing to add again behind the supernatant the identical BO liquid of 1ml and mixing, get the 20-30 piece of fertility of oocytes liquid that contains that 50ul is added to 50ul and (contain 10mg/ml BSA, the BO liquid of fatty acids not) after cultivating 5 hours altogether in, be transferred to and continue among the vitro culture liquid CR1aa to cultivate 5 to 7 days, treat that development of fertilized ova observes with fluorescent microscope to mulberry fruit to blastula stage, the embryo of containing green fluorescence shows that this embryo contains Y chromosome, be male embryo, otherwise be female embryo.
Claims (13)
1. modified cell line of male mammal, its Y chromosome is integrated with the labelled protein gene; This cell line of male mammal is to obtain with the method that may further comprise the steps:
1) will contain in the somatocyte of labelled protein expression carrier importing boar;
2) distinguished sequence with described boar Y chromosome is a probe, carries out fluorescence in situ hybridization with somatocyte in the step 1), and screening obtains having the boar somatocyte of the Y chromosome of being modified by described labelled protein;
Described Mammals is not the people.
2. cell line of male mammal according to claim 1 is characterized in that: described labelled protein gene is fluorescence protein gene, beta-galactosidase gene, luciferase gene or GRD beta-glucuronidase gene.
3. cell line of male mammal according to claim 2 is characterized in that: described fluorescence protein gene is for strengthening green fluorescence protein gene or green fluorescence protein gene.
4. according to claim 1 or 2 or 3 described cell line of male mammal, it is characterized in that: described Y chromosome also is integrated with selectable marker gene.
5. cell line of male mammal according to claim 4 is characterized in that: described selectable marker gene is a neomycin resistance gene.
6. method that obtains modified cell line of male mammal may further comprise the steps:
1) will contain in the somatocyte of labelled protein expression carrier importing boar;
2) distinguished sequence with described boar Y chromosome is a probe, carries out fluorescence in situ hybridization with somatocyte in the step 1), and screening obtains having the boar somatocyte of the Y chromosome of being modified by described labelled protein;
Described Mammals is not the people.
7. method according to claim 6 is characterized in that: the expression vector in the described step 1) also contains selectable marker gene.
8. method according to claim 7 is characterized in that: described selectable marker gene is a neomycin resistance gene.
9. according to claim 6 or 7 or 8 described methods, it is characterized in that: described labelled protein gene is fluorescence protein gene, beta-galactosidase gene, luciferase gene or GRD beta-glucuronidase gene; Described expression vector also contains the IRES sequence.
10. according to claim 6 or 7 or 8 described methods, it is characterized in that: the probe that carries out fluorescence in situ hybridization with somatocyte in the described step 1) also has labelled protein gene fragment and/or selectable marker gene fragment.
11. according to claim 6 or 7 or 8 described methods, it is characterized in that: described boar is male domestic animal or mouse.
12. method according to claim 11 is characterized in that: described male domestic animal is bull, ram, buck or boar.
13. a method of cultivating boar, be with claim 1,2,3, the 4 or 5 described boar somatocyte of modifying through marker gene as for the nucleome cell, obtain boar through the somatic cell clone method; Described Mammals is not the people.
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| CN 200410074503 CN1745626B (en) | 2004-09-06 | 2004-09-06 | Modified cell line of male mammal, its production and use thereof |
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| CN103299950A (en) * | 2012-03-07 | 2013-09-18 | 苏州西山中科实验动物有限公司 | Method for establishing machin hyperlipidemia and atherosclerosis model |
| CN104450673B (en) * | 2014-11-14 | 2017-07-21 | 中国农业大学 | A kind of Y chromosome method of modifying and its application |
| CN104388560B (en) * | 2014-11-14 | 2017-04-12 | 中国农业大学 | Method for marking Y chromosome and application thereof |
| CN114015705A (en) * | 2021-11-28 | 2022-02-08 | 华中科技大学同济医学院附属协和医院 | Sex selection method for mouse in-vitro fertilization breeding |
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