CN101851639A - Locating and integrating carrier for human gdnf gene on cow beta-casein locus and application thereof - Google Patents

Abstract

The invention provides a locating and integrating carrier for human gdnf gene on cow beta-casein locus, comprising a positive screening gene of which two ends are respectively provided with a recombinase recognition sequence, the other end of the recombinase recognition sequence is respectively connected with the 5' isogeny arm and the 3' isogeny arm of the cow beta-casein gene; the human gdnf gene is positioned on the downstream of the 5' isogeny arm in which a promoter used for starting positive screening gene expression is arranged; and the outside of the isogeny arm is provided with a negative screening gene. The invention also provides the application of the carrier in preparing a reactor cow breast bioreactor for expressing human GDNF protein. The carrier preparation animal breast bioreactor provided by the invention can efficiently express the human GDNF protein factor with biologic activity and has large output, easy purification, simple equipment, environment protection, short development period and low production cost.

Description

People gdnf gene is in the positioning integration carrier and the application thereof of ox β-casein locus

Technical field

The present invention relates to animal mammary gland bioreactor, specifically, relate to people gdnf gene and efficiently express application in the bovine mammary gland bio-reactor of people's gdnf protein at the positioning integration carrier of ox β-casein locus and in preparation.

Background technology

Glial cellline-derived neurotrophic factor (glial cell line-direved neurotrophicfactor; GDNF) be the glial cell derived neurotrophic factor of brain.GDNF is a kind of secreting glycoprotein.The GDNF precursor is made of 211 amino acid, contains 9 amino acid of signalase 11 and 58 amino acid of leader (prepro), and mature polypeptide is 134 amino acid, wherein contains two N-glycosylation sites.Discover that GDNF has trophism to multiple neurone, for example:

(1) GDNF is to the effect of dopaminergic neuron

Lin etc. find that at first GDNF can promote the survival of rat midbrain dopaminergic neuron and differentiation of vitro culture specifically, and can increase the picked-up of these cells to Dopamine HCL.Experimental study has also obtained same result in the body, and promptly GDNF has the specificity trophism to midbrain dopaminergic neuron, is a kind of effective dopaminergic neuron nutritional factor.In multiple animal model for parkinsonism; for example; cut off the rat that causes the nigral cell damage with mouse and monkey, one-sided 6-OHDA and operation that neurotoxic substance MPTP causes nigral dopaminergic neuron unit to degenerate, GDNF is further confirmed the protection and the repair of dopaminergic neuron.GDNF also can obviously improve the dyskinesia symptoms such as bradykinesia, stiff and posture instability of Parkinson's model monkeys.Present achievement in research shows that GDNF is a kind of potent, the special relatively dopaminergic neuron protection factor, has demonstrated huge potential value and application prospect in Parkinsonian treatment.FDA Food and Drug Administration (FDA) has ratified the treatment research that GDNF is used for Parkinson and has entered three phase clinical stages.

(2) GDNF is to the effect of motor neuron, autonomic neuron and Sensory neurone

GDNF also is a kind of motor neuron nutritional factor, and gdnf gene transfection carrier cell is cultivated with the nutrient solution that contains brain and dynamoneure, can prolong the length of these motor neuron axons, reduces normal apoptotic.The mouse of gdnf gene disappearance, its spinal motion neurone has reduced 22%.Give GDNF after cutting off newborn rat spinal nerves and facial nerve aixs cylinder, can the neuronic degeneration of retardation motion.GDNF can promote the sympathetic neuron and the parasympathetic neuron survival of cultivating to autonomic neuron and the equally also nutritious effect of Sensory neurone.In the family that lacks gdnf gene, the differentiation of Sensory neurone is obstructed, and shows as to carry out of short duration dopamine hydroxylase expression, and the reorganization of acceptor obviously reduces with conversion.GDNF has effect equally to other neurones in the brain.It can promote the noradrenaline serotonergic neuron survival of locus coeruleus, protects these neurones to exempt from the murder by poisoning of 6-OHDA; Also can prevent death and atrophy that the damage of basal forebrain cholinergic neuron causes; Wild neuronic survival of cerebellum Pu Ken and differentiation are played an important role equally.

Recently the function that studies show that GDNF only limits to treat Parkinson's disease far from, especially noticeable is to neuropathic pain press down pain or ease pain also demonstrates magical effect, to multiple central nervous system disease, huge potential value and application prospect have been demonstrated as the treatment of apoplexy, Spinal injury, surgery brain injury, neurone degenerative disease.

For the purpose of scientific research and medicinal research, need to produce a large amount of GDNF.GDNF is a glycosylated protein, and content is very low in the humans and animals body, and extracting GDNF from animal body, to be used for clinical study be unpractical.Though adopt protokaryon fermentation using bacteria method producer gene recombinant protein easy, can not glycosylation, the protein of generation does not have physiologically active or activity very low, does not have pharmic function.External the physiologically active of product is good, but yields poorly with eukaryotic cell engineering method production GDNF, and the cost height costs an arm and a leg, and product is suitable for scientific research, is not suitable for working as drug use.

Animal mammary gland bioreactor is based on the transgenic technology platform, foreign gene imported in the animal gene group and localization and expression in animal's mammary gland, utilize animal's mammary gland can natural, efficiently synthesize the proteic ability of justacrine, the albumen mass-energy of institute's output is Natively glycosylated, and have production efficiency height, a low cost and other advantages.Thereby it is to have one of technological method of development prospect most that animal mammary gland bioreactor is known as in the world in human disease treatment and medical protein production for health care.

Use animal mammary gland bioreactor and produce the focus that pharmaceutical protein has become biological high-tech field research, yet, traditional gene random integration method is made galactophore biological reactor, the expression of exogenous gene regulation and control are subjected to the contiguous dna sequence dna influence of integration site, and expression level difference rests on lower expression level greatly and mostly.And the transgenic animal exogenous gene expression of making of the gene targeting method is not influenced by position effect, can significantly improve expression level.Therefore, making galactophore biological reactor with the gene targeting method is the optimal selection of current production medical protein.

Summary of the invention

The purpose of this invention is to provide the positioning integration carrier of a kind of people gdnf gene at ox β-casein locus.

Another object of the present invention provides the application of above-mentioned carrier in the bovine mammary gland bio-reactor of preparation expressing human gdnf protein.

In order to realize the object of the invention, a kind of people gdnf gene of the present invention is at the positioning integration carrier of ox β-casein locus, it comprises positive screening-gene, two ends at positive screening-gene have the recombinase recognition sequence, the other end of described recombinase recognition sequence links to each other with 3 ' homology arm with 5 ' homology arm of ox β-casein gene respectively, people gdnf gene is positioned at 5 ' homology arm downstream, the inside of described 5 ' homology arm has the positive screening-gene expression promoter of startup, also has negative screening-gene in the outside of described homology arm.

Aforesaid carrier, wherein said positive screening-gene is the gene of coding neomycin phosphotransferase.

Aforesaid carrier, wherein said negative screening-gene are two negative screening factors, are respectively the red fluorescent mark gene of HSV-tk gene and DsRed2.

Aforesaid carrier, wherein said recombinase recognition sequence is the Cre enzyme recognition sequence.

Aforesaid carrier, the primer of the described ox β-casein gene 5 ' homology arm that wherein increases be upstream primer 5 '-GACGTCGACAAAACTTCCGTGTGTCCCAGC-3 ' and downstream primer 5 '-GGCCTCGAGCTCCTGGGAATGGGAAGATGA-3 '.

Aforesaid carrier, the primer of the described ox β-casein gene 3 ' homology arm that wherein increases be upstream primer 5 '-AAAGGATCCAGCAACAGACTAACAAGAAGG-3 ' and downstream primer 5 '-CTCAGGATCCAAACATCGGCTTACTTG-3 '.

Aforesaid carrier, the primer of the described people gdnf gene that wherein increases be upstream primer 5 '-GACCTCGAGATGAAGTTATGGGATGTCGTGGCTGTC-3 ' and downstream primer 5 '-GAGCTCGAGTCAGATACATCCACACCTTTTAG-3 '.

Aforesaid carrier, the downstream of wherein said people gdnf gene is connected with SV40 polyA sequence.

Described people gdnf gene is in the application of positioning integration carrier in gene targeting of ox β-casein base locus.

Described people gdnf gene is in the application of positioning integration carrier in the bovine mammary gland bio-reactor of preparation expressing human gdnf protein of ox β-casein locus.

By technique scheme, the present invention has following advantage and beneficial effect at least:

(1) the present invention adopts the method for homologous recombination, with the downstream of goal gene gdnf positioning integration to ox β-casein gene promoter, makes goal gene efficiently express by ox β-casein gene natural promoter.

(2) galactophore biological reactor that adopts the inventive method to prepare can efficiently express in mammary epithelial cell and have biologic activity, glycosylated people's gdnf protein factor.

(3) adopt the red fluorescent mark gene of DsRed2 as one of negative screening factor of practicing shooting, help improving the screening efficiency of cell targeting.

(4) the present invention utilizes livestock animals galactophore biological reactor production pharmaceutical protein to have the following advantages: mammary gland can constantly be secreted milk, and long-term collection can not damage animal; Physiological activity influence to animal is little, and pharmaceutical protein is limited in the mammary gland and produces, and is secreted in the milk, can not enter blood, can the normal physiological activity of transgenic animal not impacted; The biological activity height, mammary gland of domestic animal can carry out a series of translation post-treatment such as glycosylation, esterification, phosphorylation and formation polymer to expressed protein, and product is near natural extract, and is active high, is difficult for developing immunity to drugs; Output is big, easily purifies; Equipment is simple, non-environmental-pollution; Construction cycle is short; Production cost is low.

(5) to prepare galactophore biological reactor different with traditional gene random integration method, and its expression of exogenous gene regulation and control are vulnerable to the contiguous dna sequence dna influence of integration site, and expression level difference rests on lower level greatly and mostly; The present invention adopts the transgenic animal foreign gene localization and expression of gene targeting method preparation not influenced by position effect, can significantly improve expression level.

Description of drawings

Fig. 1 is a gdnf PCR product gel electrophorogram of the present invention, and 1 is dna molecular amount standard, 100bp DNA Ladder Marker, and 2 is people gdnf cDNA PCR product;

Fig. 2 is ox β of the present invention-casein gene 5 ' homology arm PCR product electrophorogram, and 1 is dna molecular amount standard, 500bp DNA Ladder Marker, and 2 is 5 ' homology arm PCR product;

Fig. 3 is ox β of the present invention-casein gene 3 ' homology arm PCR product electrophorogram, and 1 is dna molecular amount standard, and λ DNA/EcoR I, 2-4 are 3 ' homology arm PCR product;

Fig. 4 is the structural representation of gene targeting carrier pNRTCNbG of the present invention;

Fig. 5 is the tolerance experimental result synoptic diagram of inventor's breast tumor epithelial cell Bcap-37 to different concns G418;

The transgenic cell of Fig. 6 for examining under a microscope behind the linearized vector pNRTCNbG transfection Bcap-37 cell of the present invention, a is the phase microscope observations, b is the fluorescence microscope result;

Fig. 7 is the pcr analysis of transgenic cell genomic dna of the present invention, and 1 is dna molecular amount standard, λ DNA/HindIII+EcoR I, and 2 is plasmid pNTRCNbG positive control, and 3 is the normal cell negative control, and 4 is transfectional cell Bcap-37;

Fig. 8 analyzes transcribing of transgenosis Bcap-37 cell recombinant human gdnf for RT-PCR of the present invention, 1 is dna molecular amount standard DL2000 DNA Marker, 2-4 is with inducing culture 24hr, 48hr and 72hr non-transgenic cell total rna are the PCR of template, 5-7 is with inducing culture 24hr, 48hr and the total RNA of 72hr transgenic cell are the PCR of template, 8-10 is inducing culture 24hr, the RT-PCR of 48hr and 72hr non-transgenic cell, 11-13 is inducing culture 24hr, the RT-PCR of 48hr and 72hr transgenic cell;

Fig. 9 is the secretion of Western Blot detection reorganization gdnf protein of the present invention, and M is albumen Marker, and 1 is the normal Bcap-37 cell conditioned medium liquid of inducing culture 48hr, and 2 is the transgenosis Bcap-37 cell conditioned medium liquid of inducing culture 48hr;

Figure 10 detects figure for the PCR of the gene targeting clone blastaea that the present invention obtains, and 1 is λ DNA/HindIII+EcoR I, and 2 is H ₂O (negative control), 3 is lonely female blastaea (negative control), and 4 and 5 are gene targeting clone blastaea, and 6 is pNRTCNbG (positive control).

Embodiment

Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.

The preparation of the bovine mammary gland bio-reactor efficient expression vector of embodiment 1 people gdnf gene targeting β-casein locus

1 materials and methods

1.1 material

1.1.1 bacterial strain and plasmid

Bacillus coli DH 5 alpha is available from precious biotechnology (Dalian) company limited; Plasmid pCMV-Red inserts the CMV promotor by pDsRed2 (Clontech) and derives in BamH I site; Plasmid pPGK-neoLoxP is so kind as to give by Washington, DC medical college; Plasmid pUMVC1-tk is available from Michiganite U.S.A university carrier center.

1.1.2 main agents and test kit

Plasmid extraction kit, a small amount of RNA extract test kit, DNA sepharose recovery test kit and DNA purification kit available from QIAGEN, Shanghai China Shun biotechnology company limited and TIANGEN; Taq Plus, Pfu archaeal dna polymerase are available from TIANGEN; ExTaq archaeal dna polymerase, T4DNA polysaccharase, T4DNA ligase enzyme, M-MLV ThermoScript II, oligo (dT) and restriction enzyme are the TAKARA product.The PCR primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.

1.1.3 key instrument equipment

Electric heating constant temperature water isolation type incubator (DDHZ-300), constant temperature water bath case (Shanghai one perseverance), vibrator (Haining instrument plant), vapour is bathed vibration shaking table (THZ-C), horizontal strip electrophoresis groove (Beijing Liu Yichang), gel imaging instrument (BIO-RAD), supercentrifuge (EPPENDORF), high speed low temperature centrifugal machine (BECKMAN), large-scale low-temperature whizzer (BECKMAN) Bechtop (AIRTECH), Ultralow Temperature Freezer (SANYO), gene electroporation (BIO-RAD), nucleic acid-protein determinator (BIO-RAD), high pressure autosterilization pot (HIRAYAMA).

1.1.4 solution preparation

(1) liquid LB substratum

Yeast extract 5g, tryptone 10g, NaCl 10g is dissolved in the 800mL deionized water, transfers pH to 7.0, is settled to 1000mL with deionized water at last, autoclaving, 4 ℃ of preservations are standby;

(2) solid LB substratum

Yeast extract 5g, tryptone 10g, NaCl 10g, agar 15g is dissolved in the 800mL deionized water, transfers pH to 7.0, is settled to 1000mL with deionized water at last, autoclaving, 4 ℃ of preservations are standby;

(3) SOB substratum

Tryptones 20g, yeast extract 5g, NaCl 0.5g adds deionized water to 800mL, stirring and dissolving adds 250mmol/L KCl 10ml then, transfers pH to 7.4, be settled to cumulative volume 1L with deionized water at last, autoclaving adds 5mL through autoclaved 2MMgCl before using ₂

(4) the total DNA extraction buffer of cell

10mmol/L TrisCl (pH8.0), 0.1mmol/L EDTA (pH8.0), 20 μ g/mL trypsinase, 0.5%SDS;

(5) 50 * TAE damping fluids

242g Tris alkali, 56.7mL glacial acetic acid, 100mL 0.5M EDTA (pH8.0).

1.2 method

1.2.1RT-PCR amplification gdnf gene

Get 4 monthly age human fetal renal tissue 30mg; use RNA available from Shanghai China Shun's biotechnology company limited to extract test kit in a small amount and extract total RNA; and be synthetic cDNA first chain of primer with oligo (dT); then as template; carry out pcr amplification with people gdnf gene 5 ' end and 3 ' two primers of end respectively, reaction conditions is: 94 ℃ of sex change 45s, 58 ℃ of renaturation 45s; 72 ℃ are extended 1min, totally 35 circulations.Gdnf PCR primer is upstream primer G1:5 '-GAC CTCGAGATGAAGTTATGGGATGTCGTGGCTGTC-3 ', downstream primer G2:5 ' GAG CTCGAGTCAGATACATCCACACCTTTTAG-3 '; Upstream and downstream primer 5 ' end is introduced Xho I restriction enzyme site respectively.

1.2.25 the pcr amplification of ' homology arm and 3 ' homology arm

Ox β-casein gene order (M55188) design 5 ' homology arm delivered according to Bonsing etc. and the pcr amplification primer of 3 ' homology arm make product length be respectively 2264bp and 5744bp.5 ' homology arm PCR upstream primer CNb1:5 '-GAC GTCGACAAAACTTCCGTGTGTCCCAGC-3 ', downstream primer CNb2:5 '-GGC CTCGAGCTCCTGGGAATGGGAAGATGA-3 ', primer two ends, upstream and downstream are introduced Sal I and Xho I restriction enzyme site respectively; 3 ' homology arm PCR upstream primer CNb3:5 '-AAA GGATCCAGCAACAGACTAACAAGAAGG-3 ', downstream primer CNb4:5 '-CTCA GGATCCAAACATCGGCTTACTTG-3 ', BamH I restriction enzyme site is introduced at primer two ends, upstream and downstream respectively.With the bovine fetal fibroblast is material, extracts genomic dna, is template with the genomic dna, with high-fidelity ExTaq archaeal dna polymerase amplification 5 ' homology arm and 3 ' homology arm.The PCR reaction conditions of amplification 5 ' homology arm is: 94 ℃ of sex change 45s, and 61.5 ℃ of renaturation 45s, 72 ℃ are extended 2.5min, totally 35 circulations.The PCR reaction conditions of amplification 3 ' homology arm is: 94 ℃ of sex change 45s, and 58 ℃ of renaturation 45s, 72 ℃ are extended 6min, totally 35 circulations.

1.2.3 the structure of gene targeting carrier pNRTCNbG

1.2.3.1 the insertion of goal gene gdnf transcription termination signal sequence

With Not I, Ssp I digested plasmid pCMV-Red, reclaim the SV40polyA transcription termination signal sequence of 264bp; With Not I, EcoRV digested plasmid pPGK-neoLexP, SV40polyA is cloned on the pPGK-neoLoxP carrier called after pP40.

1.2.3.2 the insertion of negative selection markers gene DsRed2

With BamH I, Ssp I digested plasmid pCMV-Red, glue reclaims the DsRed2 DsRed gene of 1.5kb; Cut the pP40 carrier with Kpn I enzyme, the T4DNA polysaccharase is mended flat cohesive end, after cutting with BamH I enzyme again, with the DsRed2 gene clone to the pP40 carrier, called after pP40R.

1.2.3.3 the insertion of negative selection markers gene HSV-tk

Is that template amplification length is the HSV-tk gene of 2837bp with high-fidelity Pfu archaeal dna polymerase with plasmid pUMVC1-tk; The PCR upstream primer: 5 '-GTT CTCGAGTGTCGGGGCTGGCTTA-3 ', downstream primer: 5 '-CCA CACGTGATAGTCCTGTCGGGTTTCGC-3 '; Primer two ends, upstream and downstream are introduced Xho I and PmaC I (being used for the targeting vector linearizing) restriction enzyme site respectively; The PCR reaction conditions is: 94 ℃ of sex change 45s, and 62 ℃ of renaturation 45s, 72 ℃ are extended 3min, totally 35 circulations.With Apa I digested plasmid pP40R, the T4DNA polysaccharase is mended flat cohesive end, and after cutting with Xho I enzyme, glue reclaims the HSV-tk gene of pcr amplification again, after cutting with Xho I enzyme, is cloned on the pP40R carrier called after pP40RT.

1.2.3.4 the insertion of 5 ' homology arm

Cut 5 ' homology arm PCR product with Sal I, Xho I enzyme, 5 ' homology arm is cloned into the Xho I site of pP40RT, called after pP40RTC.

1.2.3.5 the insertion of goal gene gdnf

Gdnf PCR product is cut the Xho I site of rear clone to the pP40RTC carrier, called after pP40RTCG with Xho I enzyme.Detect the direction of insertion of gdnf with the PCR method; The PCR upstream primer (5 '-ACTATTTC CTCATCTTCCCATTCCCAG-3 ') design is on 5 ' homology arm of carrier, and downstream primer is gdnf 3 ' end primer G2:5 '-GAG CTCGAGTCAGATACATCCACACCTTTTAG-3 '; Reaction conditions is: 94 ℃ of sex change 45s, and 62 ℃ of renaturation 45s, 72 ℃ are extended 1min, totally 35 circulations; If forward inserts the special product that will obtain 598bp, oppositely insertion does not then have the specific PCR product.

1.2.3.6 the insertion of 3 ' homology arm

3 ' homology arm PCR product is cut the BamH I site of rear clone to the pP40RTCG carrier with BamH I enzyme.Detect the direction of insertion of 3 ' homology arm with the PCR method; The PCR upstream primer: 5 '-TGCCTCTGAATGAACACTATCTTACC-3 ' (being positioned on 3 ' homology arm), downstream primer: 5 '-GTCTGAGTAGGTGTCATTCTATTCTGG-3 ' (being positioned on 3 ' homology arm 5 ' end outside skeleton carrier sequence); Reaction conditions is: 94 ℃ of sex change 45s, and 58 ℃ of renaturation 45s, 72 ℃ are extended 1min, totally 35 circulations; If forward inserts the special product that will obtain 829bp, oppositely insertion does not then have the specific PCR product.

1.2.4 the partial dna sequence of gene targeting carrier is measured

By carrying out dna sequence analysis to inserting fragment and skeleton carrier junction and insertion fragment with insertion fragment junction, further whether the identified gene targeting vector makes up correct.Order-checking is finished by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Sequencing primer and the position of mensuration sequence in the pNRTCNbG carrier are as shown in table 1.

Table 1 pNRTCNbG carrier part determined dna sequence position

2 results

2.1RT-PCR amplification obtains the gdnf gene

The RT-PCR amplified production detects with 1% agarose gel electrophoresis, and specific band conforms to (576bp) (as shown in Figure 1) with the expection size.The result shows, has obtained gdnf cDNA by the RT-PCR amplification.Sequencing result shows that the gdnf sequence is consistent with the sequence (AY052832) that GenBank announces.

2.2PCR amplification obtains 5 ' homology arm and 3 ' homology arm

The cow genome group DNA that extracts, the agarose gel electrophoresis with 0.7% detects, and its integrity meets the PCR requirement greater than 21kb.As template, 5 ' homology arm (as shown in Figure 2) of 2246bp and 3 ' homology arm (as shown in Figure 3) of 5744bp have been obtained by pcr amplification.

2.3 the evaluation of gene targeting carrier

2.3.1 the enzyme of recombinant plasmid pP40 is cut evaluation

With Not I, EcoR I double digestion recombinant plasmid pP40, the small segment of acquisition conforms to the expection size, shows that SV40 polyA is inserted into the desired location of skeleton carrier.

2.3.2 the enzyme of recombinant plasmid pP40R is cut evaluation

The big fragment that obtains with HindIII, BamH I double digestion recombinant plasmid pP40R equates that with pP40 single endonuclease digestion clip size small segment is about 1555bp, and it is correct to show that pP40R makes up.

2.3.3 the enzyme of recombinant plasmid pP40RT is cut evaluation

The big fragment that obtains with PmaC I, Xho I double digestion recombinant plasmid pP40RT equates that with pP40R single endonuclease digestion clip size small segment is about 2827bp, and it is correct to show that pP40RT makes up.

2.3.4 the enzyme of recombinant plasmid pP40RTC is cut evaluation

Sal I and Xho I are isocaudarner, and 5 ' homology arm is inserted into carrier Xho I site, and 5 ' end will form Taq I restriction enzyme site, and 3 ' end will form Xho I restriction enzyme site.With PmaC I, Xho I double digestion recombinant plasmid, judge the direction of insertion of 5 ' homology arm according to the big I of endonuclease bamhi.

2.3.5 the evaluation of recombinant plasmid pP40RTCG

Cut 5 recombinant plasmids with Xho I enzyme, all obtained the small segment of 564bp.PCR detects the direction of insertion of gdnf, and wherein 3 are the forward insertion, and 2 is oppositely to insert.

2.3.6 the evaluation of recombinant plasmid pNRTCNbG

Cut 2 recombinant plasmids with BamH I enzyme, all obtained the big fragment of 13kb and the small segment of 5.7kb.PCR detects the direction of insertion of 3 ' homology arm, and one of them inserts for forward, and another is oppositely to insert.Forward inserts, and is the gene targeting carrier pNRTCNbG of correct structure, and its structural representation as shown in Figure 4.

2.4 the part dna sequencing of gene targeting carrier pNRTCNbG is analyzed

By pNRTCNbG carrier part dna sequence dna sequencing result is shown, insert being connected and inserting that fragment and insertion are segmental to be connected correctly of fragment and skeleton carrier, proved that further constructed targeting vector structure is correct.

The biological function analysis of embodiment 2 people GDNF bovine mammary gland bio-reactor efficient expression vectors

The pNRTCNbG carrier is changed among HBT's epithelial cell line Bcap-37, behind hormone induction, in cell culture supernatant, detected the specifically expressing of people GDNF, verified that this expression vector has correct biological function.

1 materials and methods

1.1 material

1.1.1 key instrument equipment

Biologic cleanliness safety cabinet (BHRC-1300IIA/B3), CO ₂Incubator (BINDER), inverted phase contrast microscope (NIKON), Ultralow Temperature Freezer (SANYO), PCR instrument (BIOMETRO), gel imaging instrument (BIO-RAD), DYY-6C type electrophoresis apparatus and electrophoresis chamber (Liuyi Instruments Plant, Beijing), high speed tabletop centrifuge (EPPENDORF), the low speed desk centrifuge (is gone up Hai Anting, TGL-16G), protein electrophoresis instrument and commentaries on classics film instrument (BIO-RAD) etc.

1.1.2 living materials

HBT's epithelial cell line Bcap-37 is available from Shanghai cell institute of the Chinese Academy of Sciences.

1.1.3 main agents

RPMI-1640 substratum, G418, trypsin Trypsin), Regular Insulin (Insulin), sheep prolactin (Prolactin) and hydrocortisone (Hydrocortisone) be the Sigma product; The standard foetal calf serum is the TBD product; Taq Plus archaeal dna polymerase is available from TIANGEN; ExTaq archaeal dna polymerase, M-MLV ThermoScript II, oligo (dT) and restriction enzyme are the TAKARA product; No intracellular toxin plasmid extracts test kit and DNA purification kit in a large number available from QIAGEN; RNA extracts test kit available from Shanghai China Shun biotechnology company limited in a small amount; Rabbit GDNF polyclone is available from Santa Cruz; Two anti-(goat-anti rabbits) of alkaline phosphatase covalency coupling connection and BCIP/NBT test kit are available from doctor's moral company.

1.1.4 main solution preparation

(1)0.01M?PBS(pH7.4)

Take by weighing Na ₂HPO ₄1.1648g, NaH ₂PO ₄2H ₂O 0.2825g, NaCl 9.0g add ultrapure water and are settled to 1L, and behind the autoclaving, 4 ℃ store for future use;

(2)1×TE(pH7.6)

100mM TrisCl (pH7.6) 10mL, 10mM EDTA (pH8.0) 2mL adds ultrapure water and is settled to 1L, and behind the autoclaving, 4 ℃ store for future use;

(3) the dense liquid storage of G418 (100mg/mL)

Take by weighing G418 powder 1.0g, after adding ultrapure water 10mL dissolves fully, 0.22 μ m filter filtration sterilization ,-20 ℃ store for future use;

(4) 0.25% trypsinase/EDTA

Take by weighing trypsinase 0.5g, EDTA2H ₂O 0.084g adds ultrapure water 100mL, divides in the 15mL centrifuge tube of packing into behind the 0.22 μ m filter filtration sterilization, and-20 ℃ store for future use;

1.1.5 nutrient solution

HBT's epithelial cell nutrient solution: RPMI-1640+20%FBS

HBT's epithelial cell inducing culture liquid:

RPMI-1640+Insulin?0.4U/mL+Hydrocortisone?1μg/mL+Prolactin?4μg/mL

1.2 method

1.2.1 HBT's epithelial cell Bcap-37 detects the tolerance of G418

With the Bcap-37 cell with 5 * 10 ⁴The density of individual cells/well is inoculated in 12 orifice plates, when cell grows to 80% degree of converging, change nutrient solution, in 12 holes, add respectively simultaneously G418 to final concentration be 0 μ g/mL, 50 μ g/mL, 100 μ g/mL, 150 μ g/mL, 200 μ g/mL, 250 μ g/mL, 300 μ g/mL, 350 μ g/mL, 400 μ g/mL, 450 μ g/mL, 500 μ g/mL, 550 μ g/mL, changed liquid once in per 2 days, every day the observation of cell growing state, the whole killed times of cell in different G418 concentration cultivated in record, the most suitable concentration with the G418 concentration of killing whole cells in 7-10 days during as screening.

1.2.2 liposome mediated-method transfection HBT epithelial cell Bcap-37

Transfection the day before yesterday is with the cell of tryptic digestion logarithmic phase, by 3 * 10 ⁵The density of individual cells/well is inoculated in six orifice plates, and every hole adds 2mL and do not contain antibiotic substratum, makes it reach the degree of converging of 50-80% when transfection;

2 μ g linearizing targeting vectors are joined in the 1.5mL centrifuge tube and with serum-free medium be diluted to 100 μ L, in another centrifuge tube, add 10 μ L lipofectmine and be diluted to 100 μ L with serum-free medium, then two pipe liquid are merged, gentle mixing, room temperature is placed 30min, before the transfection, add 200 μ L serum-free mediums again, gentle mixing;

Six orifice plate cells change liquid, replace the nutrient solution of every hole 400 μ L serum-frees, add 400 μ L plasmid liposome complexes to every hole then, with shaking table mixing leniently;

After under 37 ℃, the condition of 5% saturated humidity, cultivating 6hr, changes fresh complete culture solution, continue cultivation.

1.2.3 the screening of stable transfected cells and enlarged culturing

Behind the transfection 48hr, be inoculated in the 100mm culture dish, and add G418 to final concentration 300 μ g/mL with the every porocyte of tryptic digestion, continue to cultivate, changed liquid once in per 3 days, after 8-10 days, with the cell clone of expressing red fluorescent protein separate, spread cultivation, freezing preservation is standby.

1.2.4PCR detection transfectional cell

Extract the cell genomic dna of stable transfection, with 5 ' homology arm upstream primer as sense primer, at SV40polyA sequence outside design antisense primer, PCR detects gdnf and transcribes box (5 ' arm+gdnfcDNA+SV40polyA) whether be incorporated in the cell genomic dna.The PCR reaction conditions is: 94 ℃ of sex change 45s, and 62 ℃ of renaturation 45s, 72 ℃ are extended 3min, totally 35 circulations.

1.2.5 the GDNF detection of expression of transgenic cell

1.2.5.1 the abduction delivering of transgenic cell

When positive transgenic cell grows to 80% degree of converging, change inducing culture liquid, behind difference inducing culture 24hr, 48hr, the 72hr, collecting cell and cell culture supernatant.

1.2.5.2RT-PCR analyze the transcriptional expression of gdnf

Cell with inducing culture is a material, extracts test kit with Shanghai China Shun biotechnology a small amount of RNA of company limited (no DNA), extracts total RNA to specifications, is the synthetic cDNA chain of template reverse transcription with 1.0 μ g RNA.Gdnf expression in mRNA level whether arranged for template PCR after detecting the transgenic cell inducing culture with cDNA.Gdnf primer and PCR reaction conditions are with 1.2.1 among the embodiment 1.

Simultaneously, contrast as the RT-PCR confidential reference items with the GAPDH gene.Design following PCR primer according to GAPDHmRNA sequence (NM002046):

Upstream primer: 5 '-AACGGATTTGGTCGTATTGGGCG-3 '

Downstream primer: 5 '-CAAAGGTGGAGGAGTGGGTGTCG-3 '

The PCR reaction conditions is: 94 ℃ of sex change 45s, and 60 ℃ of renaturation 45s, 72 ℃ are extended 1min, totally 35 circulations.

1.2.5.3Western Blot detects the secretion of gdnf expression product

Trichloroacetic acid method concentrating cells inducing culture supernatant liquor, the Lorry method is measured protein concentration.Get 40 μ g albumen, the 12%SDS-polyacrylamide gel electrophoresis goes on the pvdf membrane after separating, 5% skimmed milk envelope film, anti-4 ℃ of soaked overnight, two anti-incubated at room 1hr, BCIP/NBT colour developing.

2 results

2.1 HBT's epithelial cell Bcap-37 is to the tolerance of G418

Experimental result (as shown in Figure 5) shows that G418 concentration has obvious toxic action to the Bcap-37 cell when 100 μ g/mL, and along with the increase of G418 concentration, whole killed times of cell also shorten thereupon.G418 concentration is in the scope of 200-300 μ g/mL the time, cell cultivate in 7-9 days all dead.Thereby the G418 concentration of screening stable transfection foreign gene cell should be 200-300 μ g/mL, and in the present invention, the G418 concentration that is used to screen stable transfected cells is 300 μ g/mL.

2.2 the evaluation of transgenic cell

Linearized vector transfection Bcap-37 cell, G418 screening obtained to express the cell clone (shown in Fig. 6 a and b, being respectively observed transgenic cell under phase microscope and the fluorescent microscope) of red fluorescent protein after 8-10 days.Separate the red fluorescence cell that spreads cultivation, extract genomic dna, pcr amplification gdnf transcribes box (5 ' arm+gdnfcDNA+SV40polyA), non-transfected cells does not have specific band, and the red fluorocyte of stable transfection has obtained the specific band onesize with positive control (as shown in Figure 7), shows that gdnf transcribes box and is incorporated among the cellular genome.

2.3gdnf the abduction delivering of gene

2.3.1RT-PCR detect

Extracting RNA behind positive transgenic cell and non-transfected cells inducing culture 24hr, 48hr, the 72hr, is template pcr amplification gdnfcDNA with RNA, and no specific band shows that RNA is not polluted by genomic dna; With RNA is the synthetic cDNA chain of template reverse transcription, pcr amplification gdnfcDNA gene, and the transgenic cell of inducing culture 24hr, 48hr, 72hr has obtained the 558bp specific band, and negative control does not have specific band generation (as shown in Figure 8).The result shows that it is mRNA that ox β-casein gene promoter can drive gdnf cDNA genetic transcription.

2.3.2Western Blot detects

The transgenic cell supernatant liquor of inducing culture 48hr is surveyed through Western Blot, there are two special bands of about 15kDa and 18kDa to form, the 18kDa band is the corresponding glycosylation form of GDNF, and non-transfected cells does not have special band formation (as shown in Figure 9), show that transgenosis Bcap-37 cell is behind hormone induction, the gdnfcDNA gene that ox β-casein gene promoter drives can be expressed and be translated into gdnf protein, and can translate post-treatment and form glycoprotein and be secreted into the extracellular.

3 conclusions

The present invention changes mammary gland-specific expression vector in HBT's clone Bcap-37 cell over to, has obtained the transgenic cell of stable transfection.When transgenic cell grows into 80% degree of converging,, use RT-PCR and WesternBlot and detected of the expression of recombinant human gdnf gene at mRNA and protein level through prolactin, hydrocortisone and insulin-induced expression; And reorganization GDNF can carry out being secreted into the extracellular after the glycosylation.This result shows the gene targeting carrier that we make up, and gene component is reasonable, can correctly express and secrete target protein in mammary gland cell.

The embodiment 3 people gdnf gene targeting carrier stable transfection bovine fetal fibroblasts and the clone's that hits screening

Utilize linearizing targeting vector pNRTCNbG electric shock transfection bovine fetal fibroblast, transfectional cell is divided into two equal portions, add respectively after G418 and G418+GANC screening obtains resistance clone, under fluorescent microscope, get rid of red fluorescence clone, the enrichment clone that hits.The result shows that GANC is to the toxic effect of the clone cell of enrichment, and DsRed2 albumen is to the clone cell free of toxic effects of enrichment; HSV-tk and DsRed2 bioaccumulation efficiency are respectively 2 times and 4 times.Then, utilized the transfection of linearizing targeting vector pNRTCNbG electric shock 3.2 * 10 ⁷Individual bovine fetal fibroblast.After G418 screening 8-10 days, 3057 G418 resistance clones have been obtained altogether, after under fluorescent microscope, getting rid of red fluorescence resistance clone, 773 non-red fluorescence resistance clones have been obtained, identify through methods such as pcr amplification, the order-checkings of PCR product, confirmation has 5 mono-clonals to be the clone that practices shooting, and target practice efficiency is 0.65% (5/773) relatively, and absolute target practice efficiency is 1.6 * 10 ^-7(5/3.2 * 10 ⁷).

1 materials and methods

1.1 material

1.1.1 key instrument equipment

Biologic cleanliness safety cabinet (BHC-1300IIA/B3), CO ₂Incubator (BINDER), inverted phase contrast microscope (NIKON), blood cell counting plate (Qiujing Bio-Chemical Reagent Instrument Co., Ltd., Shanghai), liquid nitrogen container (CRYOGENIC EQIPMENT), Ultralow Temperature Freezer (SANYO), gene electroporation (BIO-RAD), PCR instrument (BIOMETRO), gel imaging instrument (BIO-RAD), DYY-6C type electrophoresis apparatus and electrophoresis chamber (Liuyi Instruments Plant, Beijing), high speed tabletop centrifuge (EPPENDORF), the low speed desk centrifuge (is gone up Hai Anting, TGL-16G).

1.1.2 living materials

Female bovine fetal fibroblast.

1.1.3 main agents and consumptive material

DMEM/F12, GANC, bFGF, trypsin Trypsin) available from Sigma company; The ExTaq archaeal dna polymerase, LA Taq archaeal dna polymerase, PmaC I restriction enzyme is available from TAKARA company; The standard foetal calf serum is the TBD product; T25, T75 culturing bottle, 60mm, 100mm culture dish are Corning company product; 48 porocyte culture plates, 24 porocyte culture plates, 12 porocyte culture plates, 6 porocyte culture plates are the Nunc product.

1.1.4 solution preparation

(1)2mM?GANC

5.2mg GANC is dissolved among the 10mL 0.1N HCl, is packed as every pipe 1mL ,-20 ℃ of preservations are standby;

(2) 1000 * two anti-

Penicillin 0.6993g, Streptomycin sulphate 1.0000g is dissolved in the 10mL ultrapure water, is packed as every pipe 1mL, and-20 ℃ of preservations are standby;

1.2 method

1.2.1 electroporation transfection bovine fetal fibroblast

The cell of tryptic digestion logarithmic phase, stop digestion with the DMEM/F12 that contains 10%FBS, centrifugal collecting cell, also count with the serum-free DMEM/F12 nutrient solution re-suspended cell of 1/2 stop buffer volume again, recentrifuge, making its density with serum-free DMEM/F12 nutrient solution re-suspended cell is 1 * 10 ⁷/ mL; Getting 400 μ L cell suspensions joins in the spacing 4mm electric shock cup, it is 30 μ g/mL that the linearization plasmid pNRTCNbG12 μ g that adding digests with restriction enzyme PmaC I makes its final concentration, make DNA and cell mixing at the bottom of knocking gently glass, the transfection of shocking by electricity under 550V voltage, the 50 μ F capacitive conditions, after electric shock is finished, leave standstill 1min in the electric shock tank, ice bath 2min, then with cell with 1 * 10 ⁶Density is inoculated in the 100mm culture dish, in 37 ℃, 5%CO ₂, cultivate under the saturated humidity condition.

1.2.2 the screening of monoclonal cell

The cell of 48hr after the transfection is behind the tryptic digestion, with 3 * 10 ⁵Individual cell density is inoculated in the 100mm culture dish, add G418 to final concentration 500 μ g/mL and GANC to final concentration 2 μ mol/L, or only add G418 to final concentration 500 μ g/mL, continue to cultivate, changed liquid once in per 2 days, after 8-10 days, microscopically observation of cell clone, mark the above clone of 100 cells, under fluorescent microscope, mark red fluorocyte clone and non-red fluorocyte clone then, and carry out data statistics.

1.2.3 the picking of non-red fluorescence monoclonal cell and enlarged culturing

Under aseptic condition, with homemade diameter is that the stainless steel clone cup of 8mm picks a small amount of vaseline oil, place on the non-red fluorescence monoclonal cell, add tryptic digestive juice 50 μ L, about 37 ℃ of peptic cell 3min, when cell can take off wall down when vibrating gently, add and contain serum nutrient solution 150 μ L termination digestion, blow and beat cell gently with liquid-transfering gun and make its suspension, drawing cell suspension is transferred in 48 holes, add nutrient solution to 500 μ L/ hole, nutrient solution is: DMEM/F12+10%FBS+G418300 μ g/mL+bFGF 5ng/mL (down together).In 37 ℃, 5%CO ₂, culturing cell under the saturated humidity condition.Changed liquid once in per 3 days.

The observation of cell growing state, treat that cell grows to 80% when converging, with PBS rinsing cell 2 times, add about trypsin digestion and cell 3min, stop cell dissociation with containing the serum nutrient solution, the cell suspension of 1/5 volume is gone in the 48 new holes, continue to be cultured to converge fully after, extract genomic dna and be used for the PCR method Screening and Identification clone that hits; 4/5 cell suspension goes to 24 orifice plates continuation enlarged culturing in addition.Treat that cell grows to when converging substantially, freezing preservation or continuation enlarged culturing.

1.2.4 the evaluation of target practice cell clone

With genome karyomit(e) site-directed integration sequence is template, and the PCR method detects the homologous recombination incident.Utilizing primer P1a (5 ' homology arm outside) and P2 (on the gdnf gene) is that template is carried out the PCR detection with the non-red fluorescence monoclonal cell genomic dna of anti-G418, identifies the homologous recombination clone; For the sensitivity of increase PCR detection and the accuracy of positive findings, utilize the PCR product to be template, carry out nest-type PRC with primer P1b (5 ' homology arm outside, survey in the P1a) and P2 and further detect positive cell clone; Glue reclaims positive PCR specific band, and sequencing analysis 5 ' homology arm and locus connecting zone sequence judge whether 5 ' homology arm homologous recombination has taken place.PCR primer sequence and reaction conditions are as shown in table 2.

The PCR primer that table 2 gene targeting cell clone is identified

The preparation of genomic dna template: the every hole of the cell of 48 orifice plates adds lysis buffer (the pH8.040mM Tris-HCl of 40 μ L, 0.9%TritonX-100,0.9%Nonidet P-40,0.4mg/mL Proteinase K), collect the centrifuge tube of 1.5mL after the cracking, in 65 ℃ of water-baths, digest 30min, in 95 ℃ of water-baths, handle 10min at last, make the Proteinase K inactivation.Add 2 μ L genomic dnas in the PCR reaction system of 20 μ L as template.

2 results

2.1 the bioaccumulation efficiency of targeting vector

The targeting vector that the present invention makes up is just two negative targeting vectors, is promptly just screening the factor with the conduct of neo gene, and HSV-tk and DsRed2 are as the negative screening factor.In order to study negative screening factor HSV-tk and the DsRed2 bioaccumulation efficiency to homologous recombination, we get 8 * 10 ⁶Individual cell electric shock transfection linearizing targeting vector pNRTCNbG, after the electric shock, transfectional cell is divided into two equal portions to be cultivated, a copy of it only adds G418 (500 μ g/mL) screening resistance clone, and another part adds G418 (500 μ g/mL) and GANC (2 μ mol/L) screening resistance clone, after 8-10 days, microscopically detects the resistant cell clone one by one, and carry out mark with marking pen, can see that under fluorescent microscope most of clone is red fluorescence clone, the not rubescent fluorescence of small part clone.Red fluorescence clone is the random integration clone, has the homologous recombination clone among the non-red fluorescence clone.Antagonism cell clone, red fluorescence clone, non-red fluorescence clone data are added up (table 3), calculate the bioaccumulation efficiency of targeting vector.Data analysis shows: the bioaccumulation efficiency of HSV-tk is about 2 times (397/201), and the bioaccumulation efficiency of DsRed2 is about 4 times (397/98 or 201/52), and the bioaccumulation efficiency of two negative screening factors is about 8 times (397/52 or 2 * 4).

In drug screening resistant cell clone process, we observe the resistance clone cell that screens with G418+GANC and screen the easier aging of resistance clone cell than only with G418, and the bioaccumulation efficiency of HSV-tk only is 2 times, and the bioaccumulation efficiency of DsRed2 gene is 4 times, and only need under fluorescent microscope, get rid of red fluorescence clone and just can play inrichment, need not to add the resistance medicine, to clone's toxicological harmless effect of enrichment.Therefore, in order to obtain the vigorous homologous recombination clone of growth vigor, in the process of resistance clone drug screening subsequently, we only screen with G418.

The inrichment of table 3HSV-tk and DsRed2

Annotate: DsRed2-is meant after G418 or G418+GANC screening the not resistance clone of rubescent fluorescence

2.6.2 non-red fluorescence resistant cell clone's picking and enlarged culturing

Utilized the transfection of linearizing targeting vector pNRTCNbG electric shock 3.2 * 10 ⁷Individual bovine fetal fibroblast with the G418 screening, has obtained 3057 G418 resistance clones altogether, behind the red fluorescence resistance clone of eliminating, has obtained 773 non-red fluorescence resistance clones under fluorescent microscope.Separate non-red fluorescence resistance clone with clone's cup, be inoculated into when growing to 80% degree of converging in 48 orifice plates, after tryptic digestion, 1/5 volume cell inoculation converge to 48 orifice plates continuation enlarged culturing to 80%, extract genomic dna, be used for the PCR method and identify the clone that practices shooting; The cell inoculation of 4/5 volume treats that to 24 orifice plates cell grows to when converging substantially freezing preservation in addition.In the picking and the process that goes down to posterity of resistance clone, go down to posterity all has a considerable amount of clones to lose multiplication capacity because of cell aging at every turn.But also there is the good clone of some cellular fories can enlarged culturing to 6 orifice plate, even to the culture dish of 60mm, along with the prolongation of incubation time, it is old and feeble that cell becomes gradually, shows as cell volume increases, form change, flattening, free bubbling out such as shows at phenomenon.

Clone's evaluation and analysis 2.7 hit

With P1a and P2 is primer, and PCR detects non-red fluorescence resistance clone, has 5 clonal expansions to go out the specific band of 2737bp; Carry out nest-type PRC with these 5 positive PCR products and primer P1b, P2, all amplify the purpose band of 2336bp, glue reclaims the purpose band, gives birth to worker Bioisystech Co., Ltd by Shanghai and checks order, and the result shows that 5 clones are the homologous recombination clone.

The present invention has utilized the transfection of gene targeting carrier pNRTCNbG electric shock 3.2 * 10 ⁷Individual bovine fetal fibroblast, obtained 773 non-red fluorescence resistance clones behind the positive-negative selection, identified, confirmed to have 5 clones to be the clone that practices shooting through methods such as pcr amplification, the order-checkings of PCR product, target practice efficiency is 0.65% (5/773) relatively, and absolute target practice efficiency is 1.6 * 10 ^-7(5/3.2 * 10 ⁷) (table 4).

Table 4 bovine fetal fibroblast gene targeting efficient

Annotate: the relative target practice efficiency of data representation in the bracket

The preparation of embodiment 4 people gdnf gene targetings clone blastaea

The gene targeting bovine fetal fibroblast that obtains with embodiment 3 is a nuclear donor, and enucleation oocyte is that cytosol receptor is made the transgene clone embryo, has obtained gene targeting clone blastaea.

1 materials and methods

1.1 material

1.1.1 key instrument equipment

Micromanipulator (NARISHIGE), little smelting furnace (NARISHIGE), draw pin instrument (NARISHIGE), card grinding instrument (NARISHIGE), fusion instrument (Australia, CRYOLOGIC), stereoscopic microscope (NIKON) etc.

1.1.2 living materials

Butcher the cow ovary.

1.1.3 reagent and consumptive material

The M199 substratum is the Gibco product, DMEM/F12,6-DMAP, propidium iodide (PI), Hochest33342, Unidasa (Hyaluroniidase), cytochalasin B (Cytochalasin B), D-PBS are SIGMA company product, the standard foetal calf serum is the TBD product, and all the other reagent are homemade analytical pure; The 35mm culture dish is the Corning product, and 4 well culture plates are the Nunc product, and all the other glasswares and consumptive material are homemade.

1.1.4 solution preparation

(1) Hochest33342 (1000 * storage liquid, 5mg/mL)

The 25mg pulvis all adds 50mL six and steams in the water, is distributed into every pipe 10 μ L after the dissolving, and-20 ℃ frozen standby;

(2) hyaluronic acid enzyme solution (2.5mg/mL, storage liquid)

The 100mg pulvis is dissolved in 40mL PBS (pH7.4) solution, and 4 ℃ of preservations are standby;

(3) 6-DMAP (100 * storage liquid, 200mM)

100mg 6-DMAP is dissolved in 3.06mL DMSO, is distributed into every pipe 10 μ L, and-20 ℃ frozen standby;

(4) the dense storage liquid of CCB (100 * storage liquid, 750 μ g/mL)

The 1mg powder adds 1333.3 μ L DMSO, is distributed into every pipe 20 μ L after the dissolving, and-20 ℃ frozen standby;

(5) oocyte maturation liquid

M199+10%FBS+10mM Hepes+0.1 μ g/mL 17 beta estradiols+10 μ g/mLFSH+50 μ g/mL penicillin+60 μ g/mL Streptomycin sulphates;

(6) nuclear transplantation operation liquid

M199+25mM Hepes+10%FBS+50 μ g/mL penicillin+60 μ g/mL Streptomycin sulphates;

(7) merge liquid

0.28M+0.5μM?Hepes+0.1mM?CaCl2+0.1mM?MgSO4+0.01％PVA；

(8) fetal development nutrient solution

SOFaa+BSA，SOFaa+4％FBS。

1.2 method

1.2.1 the collection of ovary ovocyte and maturation in vitro

The ox ovary that picks up from the slaughterhouse is placed physiological saline, in 2hr, transport the laboratory to after, carefully clean ovary 4-5 time with physiological saline, room temperature is placed 5-8hr.When adopting ovum, choose the ovarian follicle of diameter 2-8 millimeter, place the culture dish of 90mm with the 20mL syringe sucking-off liquor folliculi that has No. 18 syringe needles, under stereoscopic microscope, it is good to pick out form, and cumulus cell is complete, parcel is fine and close and have the ovocyte of at least three layers of cumulus cell, washes in maturation culture solution 3-4 time, 50-100 ovarian cumulus-ovocyte complex body (COCs) places 4 orifice plates that contain 1mL/ hole maturation culture solution, at 38.5 ℃, 5%CO ₂With the ripe 18hr that cultivates under the saturated humidity condition.

1.2.2 the pre-treatment of mature oocyte

The COCs that maturation is cultivated 18hr places the D-PBS that contains 0.1% Unidasa, blows and beats repeatedly to remove the cumulus cell on mature oocyte surface with 200 μ L pipettors, washes 3 times with oocyte maturation liquid again.The ovocyte of taking off cumulus cell is placed the ripe liquid droplet of 40 μ L, under stereoscopic microscope, select and discharge first polar body and the good ovocyte of the even form of kytoplasm is used for nuclear transplantation or lonely female activation.

1.2.3 the lonely female activation of bovine oocyte

1.2.3.1 the lonely female activation of ovocyte is handled

The ovocyte of select discharging first polar body places and contains 7% alcoholic acid and grow liquid and activate 7min, changes over to then in the growth liquid (SOFaa+BSA) that contains 2mM 6-DMAP, at 38.5 ℃, 5%CO ₂With cultivate 4hr under the saturated humidity condition, wash 3 times with growing liquid (SOFaa+BSA), put into growth liquid (SOFaa+BSA) droplet of 40 μ L and cultivate, activate back 36hr and check spilting of an egg rate.

1.2.3.2 the vitro culture of parthenogenetic embryo tire

The embryo who cultivates 36hr after the female activation of orphan is picked out under stereoscopic microscope in the co-culture system that spilting of an egg embryo changes the SOFaa+4%FBS+ cumulus cell over to.Grow and cultivated 7-9 days, the statistics blastocyst rate.

The somatic nuclear transplantation 1.2.4 practice shooting

1.2.4.1 the preparation of donorcells

From liquid nitrogen container, take out the freeze pipe of freezing preservation gene targeting bovine fetal fibroblast, 37 ℃ of water-bath 1-2min, after it is melted fully, join in the centrifuge tube that fills the 5mL nutrient solution, the centrifugal 5min collecting cell of 2000rpm adds 5mL nuclear transplantation operation liquid re-suspended cell again, and is centrifugal, with 50 μ L nuclear transplantation operation liquid re-suspended cell, room temperature is used for nuclear transplantation after leaving standstill 30min then.

1.2.4.2 the stoning of ripe bovine oocyte and notes nuclear

The ovocyte that will be used for the stoning operation is after the oocyte maturation nutrient solution that contains 5 μ g/mL Hochest33342 fluorescence dyes is handled 3min, wash 3 times with ripe liquid, be placed on then in the nuclear transplantation operation liquid of the cytochalasin B (CCB) that contains 7.5 μ g/mL, add an amount of off-the-shelf transgenosis nuclear donor cell again, the operation of under the micrurgy instrument, carrying out stoning and moving nuclear.Open the fluorescence excitation device during stoning earlier, grating is placed the UV place, behind the position of definite kernel, block the fluorescence light path with grating immediately, kernel removing needle with internal diameter 20-25 μ m is inhaled stoning position and first polar body, open the fluorescence light path and check whether success (fluoroscopy is no more than 5s) of stoning, select the good target practice somatocyte of form then, it is expelled to ovocyte zona pellucida after the stoning from stoning under.

1.2.4.3 the fusion of reconstructed embryo

In maturation culture solution, wash 3 times with ovocyte-donorcells complex body of annotating after nuclear is operated finishing stoning, carry out mixing operation after in maturation culture solution, recovering then to cultivate 1hr.With ovocyte to be merged-donorcells complex body balance 2min in merging liquid, changing interpole gap then over to is that 1mm covers in the integration slot that merges liquid, adjust direction with suction pipe, make ovocyte-donorcells contact surface parallel with the two poles of the earth, imposing 2 voltages is that 160V/mm, pulse duration 20 μ s, recurrent interval are the square wave electric pulse of 1s, put into M199 after finishing for every group 30 and wash 3 times, put back in the ripe liquid, put and recover in the incubator to check the fusion situation behind the 30min.

1.2.4.4 the activation of reconstructed embryo and vitro culture

The reconstructed embryo that merges placed contain 7% alcoholic acid and grow liquid and activate 7min, change over to then in the growth liquid (SOFaa+BSA) that contains 2mM 6-DMAP, at 38.5 ℃, 5%CO ₂With cultivate 4hr under the saturated humidity condition, wash 3 times with growing liquid (SOFaa+BSA), change in growth liquid (SOFaa+BSA) droplet of 40 μ L and cultivate, activate back 36hr and check spilting of an egg rate, and pick out in the co-culture system that spilting of an egg embryo changes the SOFaa+4%FBS+ cumulus cell over to.Activate the back and cultivated 7-9 days, the statistics blastocyst rate.

1.2.5 the PCR of transgene clone blastaea detects

2 of random chooses clone blastaea is put into the PCR pipe that 10 μ L TE are housed respectively, uses liquid nitrogen and 37 ℃ of water-bath multigelations 3-5 time, is template with freeze thawing liquid then,, carries out PCR and reacts as negative control with the female blastaea of orphan.1.2.1's is identical among PCR primer and reaction conditions and the embodiment 1.

2 results

2.1 the collection of ovary ovocyte and maturation in vitro

The present invention adopts suction method to reclaim 2627 pieces of ox ovarian cumulus-ovocyte complex body altogether, ripe cultivate 18hr after, the cumulus cell around the ovocyte obviously spreads., tenuigenin normal with the ovocyte form evenly and discharge first polar body as sophisticated standard, 1844 pieces of oocyte maturations are then arranged, maturing rate is 70.2%.

2.2 the lonely female activation of bovine oocyte and ectogenesis are cultivated

The ripe bovine oocyte of discharging first polar body places and contains 7% alcoholic acid and grow liquid and activate 7min, changes over to then in the growth liquid (SOFaa+BSA) that contains 2mM 6-DMAP, at 38.5 ℃, 5%CO ₂With cultivate 4hr under the saturated humidity condition, wash 3 times with growing liquid (SOFaa+BSA), it is 74.9% that the growth liquid droplet of putting into 40 μ L SOFaa+BSA is cultivated 36hr spilting of an egg rate; Spilting of an egg embryo is changed in the co-culture system of SOFaa+4%FBS+ cumulus cell, grow and cultivated 7-9 days, blastocyst rate is 24.9% (table 5).The result shows that this culture systems can support the activation of bovine oocyte and ectogenesis to cultivate well, and the nuclear transplantation reconstructed embryo can adopt this scheme to activate with ectogenesis and cultivate.

The developmental state of table 5 ox parthenogenetic embryo tire

2.3 the fusion of reconstructed embryo and ectogenesis are cultivated

This experiment is carried out stoning altogether and is annotated 186 pieces of nuclear operation ovocytes, be used for 154 pieces of electric mixing operations, 134 pieces (fusion rate 87.0%) have been merged, grow and cultivate 134 pieces, activate back 46 pieces of the spilting of an egg of 36hr (spilting of an egg rate 34.3%), grow and cultivated 7-9 days, obtain 5 pieces of blastaeas (blastaea rate 3.7%), the results are shown in Table 6.

Table 6 transgene clone embryo's developmental state

2.4 the PCR of transgene clone blastaea detects

2 pieces of transgene clone blastaeas of random choose are with the integration situation of PCR method detection foreign gene people gdnf.Clear bright single band all appears in the PCR product of 2 pieces of transgene clone blastaeas at the 576bp place as a result, conforms to the molecular weight size of expection PCR product, and the PCR product of lonely female blastaea does not then have this big or small band to occur, and the result as shown in figure 10.This result shows that the blastaea that we obtained is the transgene clone blastaea, rather than because the lonely female blastaea that forms is not thoroughly grown in the ovocyte stoning.The blastaea that is obtained detects through PCR and turns out to be the transgenosis blastaea, and the donorcells that we use is the mono-clonal target practice cell of identifying after testing, thereby infers that the transgenosis blastaea that obtains is the gene targeting blastaea.

Though above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.

Reference

Lin?LF，Doherty?DH，Lile?JD，et?al.GDNF：a?glial?cell?line-derived?neurotrophic?factor?formid-brain?dopaminergic?neurons.Science，1993，260(5111)：1130-1132

Trupp?M，Rydén?M，

H，et?al.Peripheral?expression?and?biological?activities?of?GDNF，anew?neurotrophic?factor?for?avian?and?mammalian?peripheral?neurons.J?Cell?Biol，1995，130(1)：137-148

Tomac?A，Lindqvist?E，Lin?LF，et?al.Protection?and?repair?of?the?nigrostriatal?dopaminergic?systemby?GDNF?in?vivo.Nature，1995，373：335-339

Schuchardt?A，D’agati?V，Larsson-Blomberg?L，et?al.Defects?in?the?kidney?and?enteric?nervoussystem?of?mice?lacking?the?tyrosine?kinase?receptor?Ret.Nature，1994，367：380-383

Gash?DM，Zhang?Z，Ovadia?A，et?al.Functional?recovery?in?parkinsonian?monkeys?treated?withGDNF.Nature，1996，380：252-25

Tang?XQ，Wang?Y，Han?JS，et?al.Adenovirus-mediated?GDNF?protects?cultured?motoneurons?fromglutamate?injury.Neuroreport，2001，12(14)：3073-3076

Moore?M，Klein?R，Farinas?I，et?al.Renal?and?neuronal?abnormalities?in?mice?lacking?GDNF.Nature，1996，382：76-79

Patel?NK，Bunnage M，Plaha?P，et?al.Intraputamenal?infusion?of?glial?cell?line-derivedneurotrophic?factor?in?PD：a?two-year?outcome?study.Ann?Neurol，2005，57：298-302

Sequence table

＜110〉University of the Inner Mongol

＜120〉people gdnf gene is in the positioning integration carrier and the application thereof of ox β-casein locus

<130>KHP10112268.1

<160>18

<170>PatentIn?version?3.5

 

<210>1

<211>30

<212>DNA

＜213〉artificial sequence

 

<400>1

gacgtcgaca?aaacttccgt?gtgtcccagc 30

 

<210>2

<211>30

<212>DNA

＜213〉artificial sequence

 

<400>2

ggcctcgagc?tcctgggaat?gggaagatga 30

 

<210>3

<211>30

<212>DNA

＜213〉artificial sequence

 

<400>3

aaaggatcca?gcaacagact?aacaagaagg 30

 

<210>4

<211>27

<212>DNA

＜213〉artificial sequence

 

<400>4

ctcaggatcc?aaacatcggc?ttacttg 27

 

<210>5

<211>36

<212>DNA

＜213〉artificial sequence

 

<400>5

gacctcgaga?tgaagttatg?ggatgtcgtg?gctgtc 36

 

<210>6

<211>32

<212>DNA

＜213〉artificial sequence

<400>6

gagctcgagt?cagatacatc?cacacctttt?ag 32

 

<210>7

<211>25

<212>DNA

＜213〉artificial sequence

 

<400>7

gttctcgagt?gtcggggctg?gctta 25

 

<210>8

<211>29

<212>DNA

＜213〉artificial sequence

 

<400>8

ccacacgtga?tagtcctgtc?gggtttcgc 29

 

<210>9

<211>27

<212>DNA

＜213〉artificial sequence

 

<400>9

actatttcct?catcttccca?ttcccag 27

 

<210>10

<211>32

<212>DNA

＜213〉artificial sequence

 

<400>10

gagctcgagt?cagatacatc?cacacctttt?ag 32

 

<210>11

<211>26

<212>DNA

＜213〉artificial sequence

 

<400>11

tgcctctgaa?tgaacactat?cttacc 26

 

<210>12

<211>27

<212>DNA

＜213〉artificial sequence

 

<400>12

gtctgagtag?gtgtcattct?attctgg 27

 

<210>13

<211>23

<212>DNA

＜213〉artificial sequence

 

<400>13

aacggatttg?gtcgtattgg?gcg 23

 

<210>14

<211>23

<212>DNA

＜213〉artificial sequence

 

<400>14

caaaggtgga?ggagtgggtg?tcg 23

 

<210>15

<211>32

<212>DNA

＜213〉artificial sequence

 

<400>15

ctctcttctc?acctccatct?actccttttt?cc 32

 

<210>16

<211>32

<212>DNA

＜213〉artificial sequence

 

<400>16

ctctcttctc?acctccatct?actccttttt?cc 32

 

<210>17

<211>32

<212>DNA

＜213〉artificial sequence

 

<400>17

ttaccccaag?atctcaaaga?cccaccgaat?ac 32

 

<210>18

<211>32

<212>DNA

＜213〉artificial sequence

 

<400>18

ctctcttctc?acctccatct?actccttttt?cc 32

Claims (10)

1. people gdnf gene is at the positioning integration carrier of ox β-casein locus, it comprises positive screening-gene, two ends at positive screening-gene have the recombinase recognition sequence, the other end of described recombinase recognition sequence links to each other with 3 ' homology arm with 5 ' homology arm of ox β-casein gene respectively, people gdnf gene is positioned at 5 ' homology arm downstream, the inside of described 5 ' homology arm has the positive screening-gene expression promoter of startup, also has negative screening-gene in the outside of described homology arm.

2. carrier according to claim 1 is characterized in that, described positive screening-gene is the gene of coding neomycin phosphotransferase.

3. carrier according to claim 1 is characterized in that, described negative screening-gene is the red fluorescent mark gene of HSV-tk gene and DsRed2.

4. carrier according to claim 1 is characterized in that, described recombinase recognition sequence is the Cre enzyme recognition sequence.

5. according to each described carrier of claim 1-4, it is characterized in that, the primer of the described ox β-casein gene 5 ' homology arm that increases be upstream primer 5 '-GACGTCGACAAAACTTCCGTGTGTCCCAGC-3 ' and downstream primer 5 '-GGCCTCGAGCTCCTGGGAATGGGAAGATGA-3 '.

6. carrier according to claim 5, it is characterized in that, the primer of the described ox β-casein gene 3 ' homology arm that increases be upstream primer 5 '-AAAGGATCCAGCAACAGACTAACAAGAAGG-3 ' and downstream primer 5 '-CTCAGGATCCAAACATCGGCTTACTTG-3 '.

7. carrier according to claim 6, it is characterized in that, the primer of the described people gdnf gene that increases be upstream primer 5 '-GACCTCGAGATGAAGTTATGGGATGTCGTGGCTGTC-3 ' and downstream primer 5 '-GAGCTCGAGTCAGATACATCCACACCTTTTAG-3 '.

8. carrier according to claim 7 is characterized in that, the downstream of described people gdnf gene is connected with the SV40polyA sequence.

9. each described carrier application in gene targeting of claim 1-8.

10. the application of each described carrier of claim 1-8 in the bovine mammary gland bio-reactor of preparation expressing human gdnf protein.

Images