CN102978202A - Over-expression vector for muscle specific expression of pig IGF1 gene - Google Patents
Over-expression vector for muscle specific expression of pig IGF1 gene Download PDFInfo
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Abstract
The invention provides a secure expression vector for specific over-expression of pig endogenous gene IGF1 (C1 : Ea) in pig skeletal muscle. The vector comprises an expression cassette composed of the pig endogenous sequence sk-alpha-actinPromoter/IGF1 (C1 : Ea)/sk-alpha-actin3'UTR/SV40 enhancer in series. The expression vector verifies on a cell scale that the skeletal muscle specific sk-alpha-actin promoter can be expressed efficiently in myoblasts, and expressed at lower level in PET cells having myogenic differentiation potential, but not expressed in other type cells. By using the constructed vector for preparing transgenic mice, the detected mRNA expression level and protein expression level of the IGF1 gene in the skeletal muscle of the transgenic mice via quantification PCR and WesterBlot are 15 times and 3.5 times that of the wild mice respectively; and the IGF1 gene can be only expressed in the skeletal muscle efficiently.
Description
Technical field
The present invention relates to the over-express vector of a kind of safety type skeletal muscle specifically expressing Swine insulin-like growth factor 1 (insulin-like growth factor1, IGF1) gene.
Background technology
In research gene function and transgenic animal production process, people usually realize by carrier for expression of eukaryon, for example the pcDNA3.0 serial carrier.Traditional method often is connected to viral promotors (such as the CMV promotor) or heterology strong promoter downstream by molecular biology method with the goal gene fragment, to instruct exogenous gene expression.The method has in the research of genetic expression and function extremely widely to be used, but many research has exposed the one side of this means deficiency: the heterology gene order imports the disorder that the host often causes genome sequence, endogenous gene expression is unusual, even changes the intrinsic feature phenotype of host; Usually cause that also people are to the arguement of transgenosis ethics problem.In addition, viral promoter subsequence or instruct plasmid DNA the introducing that protokaryon holds together remaining sequence such as to copy easily causes the Transgene-safty problem, must could drop into through long-term Biosafety evaluation practical application, and is not only time-consuming but also expend financial resources.Simultaneously, ubiquity is crossed expression alien gene and is often caused the unusual of body growth etc.Therefore, when utilizing transgenic technology to carry out the animal genetic modification, the native gene sequence of same species, and the specificity of tissue expression is the basis that guarantees the transgenic animal security.
Summary of the invention
The invention provides the expression framework of the special sk-α of a boar endogenous bone bone flesh-actin promoters driven, and the special expression pig IGF1 genetic safety type expression vector of crossing of Animal muscles that contains this expression framework.
The expression cassette of the special sk-α of pig endogenous bone flesh of the present invention-actin promoters driven IGF1 gene is by sk-α-actin promotor (sk-α-actin Promoter), the pig IGF1(C1:Ea of successively series connection) gene, sk-α-actin 3 ' UTR and SV40 enhanser form; This expression cassette sequence is shown in SEQ ID No.1.
The special expression pig IGF1 genetic safety type expression vector of crossing of skeletal muscle of the present invention contains the above-mentioned special sk-α of pig endogenous bone flesh-actin promoters driven expression cassette, is to be cloned among the transposon carrier pT2-HB by the EcoRV restriction enzyme site to obtain; The sequence of this expression vector is shown in SEQ ID No.2.
The expression cassette of the special sk-α of the endogenous of pig described in the present invention bone flesh-actin promoters driven IGF1 gene is by sk-α-actin promotor, the pig IGF1(C1:Ea of successively series connection) gene, sk-α-actin 3 ' UTR and SV40 enhanser form; The endogenous sk-α of described pig-actin promotor is 2602 nucleotide sequences of cloning and sequencing; Described sk-α-actin 3 ' UTR is 751 nucleotide sequences of cloning and sequencing; Described pig IGF1(C1:Ea) gene C DS total length is 393 nucleotide sequences, have GENBANK number for NM 214256.1 from 5 ' end 13-405 position nucleotide sequence; Described SV40 enhanser is 242 nucleotide sequences, obtains from+1 2201-2442 position nucleotide sequence clone from the pGL3-control carrier.
Described expression vector security is embodied in the vector expression controlling element and goal gene is all cloned from the pig endogenous sequence, and transposon carrier sequence is peculiar by vertebrates, and the nucleotide sequence of expression vector is seen sequence table.
The special safety type expression vector of crossing expression pig IGF1 (C1:Ea) gene of skeletal muscle among the present invention, verified that at cell levels the special sk-α of skeletal muscle-actin promotor can efficiently express in sarcoplast, low expression level and in the other types cell, not expressing only in having the PEF cell of myogenic differentiation potential; The carrier that makes up among the present invention prepares transgenic mice, detect the mRNA of IGF1 gene in the transgenic mice skeletal muscle and protein expression level by quantitative PCR and Western Blot and be respectively 15 times and 3.5 times of wild-type mice, and only in skeletal muscle, efficiently express, for the transgenic pig model for preparing safety, skeletal muscle specifically expressing goal gene provides platform.
Description of drawings
Fig. 1: pcr amplification sk-α-actin Promoter product electrophorogram;
Fig. 2: pcr amplification sk-α-actin 3 ' UTR product electrophorogram;
Fig. 3: pcr amplification pig IGF1 (C1:Ea) cDNA product electrophorogram;
Fig. 4: pcr amplification SV40Enhancer product electrophorogram;
Fig. 5: expression vector element connection diagram;
Fig. 6: pT2/ α-actin-IGF1 plasmid enzyme restriction product electrophorogram;
Fig. 7: pT2/ α-actin-IGF1 plasmid map;
Fig. 8: pT2/ α-actin-△
IGF1Skeleton driven reporter gene GFP organizes cells at each;
Fig. 9: pIGF1 transgenic mouse PCR detects foreign gene;
Figure 10: pIGF1 transgenic mouse SOUTHERN BLOT detects foreign gene;
Figure 11: fluorescent quantitation detects the expression of IGF1 gene in the transgenic mice histoorgan;
Figure 12: Western Blot detects the expression of IGF1 gene in the transgenic mice histoorgan;
Figure 13: Western Blot detects the expression of IGF1 gene in transgenic mice skeletal muscle.
Embodiment
The experimental technique of mentioning among the following embodiment is ordinary method if no special instructions.
Example I, skeletal muscle specifically expressing pig IGF1 Vector construction
1, the acquisition of specific expression carrier basic building block
(1) preparation of pig sk-α-actin Promoter specific fragment
Take the landrace genomic dna as template, by the special sk-α of the primer amplification skeletal muscle of the amplification sk-α that enumerates in the table 1-actin Promoter-actin promoter fragment.Reaction system is 50 μ l, 5 μ L, 10 * Buffer, 4 μ L 2.5mM dNTP, 1 μ L, 20 μ M primer sk-α-actin Promoter F, 1 μ L, 20 μ M primer sk-α-actin Promoter R(primer sequences see Table 1), 0.5 μ L5 U/ μ L high-fidelity LA Tag polysaccharase, 2 μ l landrace genomic dna templates add ultrapure water to 50 μ L(high-fidelity enzyme available from TaKaRa Code:DR002A).The pcr amplification program: 95 ℃ of 10s, 60 ℃ of 3min circulate 35 times.Pcr amplification product detects (as shown in Figure 1) with 1% agarose gel electrophoresis.
The PCR product reclaims purifying (operation steps is seen the test kit explanation) with QIAGEN sepharose test kit, purified product is connected into PCR cloning vector pMD19-T Vector(TaKaRa Code:D102A), the step operation transforms DH5 α competence intestinal bacteria (available from the full formula in Beijing King Company, article No. CD201) on request; Bacterium liquid is coated with agarose gel plate, overnight incubation in 37 ℃ of incubators; Choose single colony inoculation and cultivate in liquid LB substratum, part bacterium liquid send company's order-checking (invitrogen Beijing Company).Sequencing result shows the recombinant vectors that contains sk-α-actin Promoter, and it is for subsequent use to extract plasmid.
(2) preparation of pig sk-α-actin 3 ' UTR specific fragment
Take the landrace genomic dna as template, by special sk-α-actin 3 ' the UTR fragment of the primer amplification skeletal muscle of the amplification sk-α-actin 3 ' UTR that enumerates in the table 1.Reaction system is 50 μ l, 5 μ L, 10 * Buffer, 8 μ L 2.5mM dNTP, 1 μ L, 20 μ M primer sk-α-actin, 3 ' UTR F, 1 μ L, 20 μ M primer sk-α-actin, 3 ' UTR R (sequence sees Table 1), 0.5 μ L5U/ μ L high-fidelity LA Tag polysaccharase, 2 μ l landrace genomic dna templates add ultrapure water to 50 μ L(high-fidelity enzyme available from TaKaRaCode:DR002A).The pcr amplification program: 95 ℃ of 10s, 60 ℃ of 1min circulate 35 times.Pcr amplification product detects (as shown in Figure 2) with 1% agarose gel electrophoresis.
The PCR product reclaims purifying (operation steps is seen the test kit explanation) with QIAGEN sepharose test kit, purified product is connected into PCR cloning vector pMD19-T Vector(TaKaRa Code:D102A), the step operation transforms DH5 α competence intestinal bacteria (available from the full formula in Beijing King Company, article No. CD201) on request; Bacterium liquid is coated with agarose gel plate, overnight incubation in 37 ℃ of incubators; Choose single colony inoculation and cultivate in liquid LB substratum, part bacterium liquid send company's order-checking (invitrogen Beijing Company).Sequencing result shows the recombinant vectors that contains sk-α-actin 3 ' UTR, and it is for subsequent use to extract plasmid.
(3) clone of pig IGF1 (C1:Ea) cDNA
Take landrace liver organization cDNA as template, by the IGF1CDS fragment of the special spliceosome form of primer amplification I class Ea of the amplification IGF1 (C1:Ea) that enumerates in the table 1 (GENBANK accession number be NM 214256.1 from 5 ' end 13-405 position nucleotide sequence).Reaction system is 50 μ l, 5 μ L, 10 * Buffer, 4 μ L 2.5mM dNTP, 1 μ L, 20 μ M primer I GF1 (C1:Ea) F, 1 μ L, 20 μ M primer I GF1 (C1:Ea) R(primer sequences see Table 1), 0.5 μ L5U/ μ L high-fidelity LA Tag polysaccharase, 2 μ l landrace liver organization cDNA templates add ultrapure water to 50 μ L(high-fidelity enzyme available from TaKaRa Code:DR002A).The pcr amplification program: 95 ℃ of 10s, 60 ℃ of 1min circulate 35 times.Pcr amplification product detects (as shown in Figure 3) with 1.5% agarose gel electrophoresis.
The PCR product reclaims purifying (operation steps is seen the test kit explanation) with QIAGEN sepharose test kit, purified product is connected into PCR cloning vector pMD19-T Vector(TaKaRa Code:D102A), the step operation transforms DH5 α competence intestinal bacteria (available from the full formula in Beijing King Company, article No. CD201) on request; Bacterium liquid is coated with agarose gel plate, overnight incubation in 37 ℃ of incubators; Choose single colony inoculation and cultivate in liquid LB substratum, part bacterium liquid send company's order-checking (invitrogen Beijing Company).Sequencing result shows the recombinant vectors that contains IGF1 (C1:Ea), and it is for subsequent use to extract plasmid.
(4) preparation of SV40Enhancer specific fragment
Take pGL3-control carrier (Promega Code:E1741) as template, by the special eukaryotic expression enhanser fragment of the primer amplification of the amplification SV40 Enhancer that enumerates in the table 1 (the pGL3-control carrier is from+1 2201-2442 position nucleotide sequence).Reaction system is 50 μ l, 5 μ L, 10 * Buffer, 4 μ L 2.5mM dNTP, 1 μ L, 20 μ M primer SV40 EnhancerF, 1 μ L, 20 μ M primer SV40 Enhancer R(primer sequences see Table 1), 0.5 μ L5 U/ μ L high-fidelity LA Tag polysaccharase, 20ng pGL3-control carrier is template, adds ultrapure water to 50 μ L(high-fidelity enzyme available from TaKaRa Code:DR002A).The pcr amplification program: 95 ℃ of 10s, 60 ℃ of 30s circulate 35 times.Pcr amplification product detects (as shown in Figure 4) with 1.5% agarose gel electrophoresis.
The PCR product reclaims purifying (operation steps is seen the test kit explanation) with QIAGEN sepharose test kit, purified product is connected into PCR cloning vector pMD19-T Vector(TaKaRa Code:D102A), the step operation transforms DH5 α competence intestinal bacteria (available from the full formula in Beijing King Company, article No. CD201) on request; Bacterium liquid is coated with agarose gel plate, overnight incubation in 37 ℃ of incubators; Choose single colony inoculation and cultivate in liquid LB substratum, part bacterium liquid send company's order-checking (invitrogen Beijing Company).Sequencing result shows the recombinant vectors that contains SV40Enhancer, and it is for subsequent use to extract plasmid.
2, the structure of pT2/ α-actin-IGF1 specific expression carrier
(1) structure of non-transposon expression vector pA-actin-IGF1
Take pGL3-control vector(Promega Code:E1741) as skeleton carrier, with the KpnI/MluI double digestion sk-α-actin Promoter is downcut from clone T carrier, be connected into skeleton carrier KpnI/MluI restriction enzyme site place, be built into the pGL3-1 intermediate carrier; Use again the NcoI/HpaI double digestion that sk-α-actin 3 ' UTR/SV40 enhancer fragment is downcut from clone T carrier, connect into the NcoI/HpaI restriction enzyme site place of intermediate carrier pGL3-1, be built into the pGL3-2 intermediate carrier; Then with the EcoRI/NcoI double digestion IGF1 is downcut from clone T carrier, be connected into the EcoRI/NcoI restriction enzyme site place of intermediate carrier pGL3-2, be built into the pA-actin-IGF1 expression vector.(ligase enzyme is available from Promega Code:M1804)
(2) structure of pT2/ α-actin-IGF1 expression vector
With restriction endonuclease EcoRV will comprise that sk-α-actin Promoter/IGF1 (C1:Ea)/sk-α-actin 3 ' UTR/SV40 enhancer is composed in series expression cassette (Fig. 5) (called after α-actin-IGF1) cut out from the pA-actin-IGF1 expression vector uses
QIAGEN sepharose test kit reclaims purifying (operation steps is seen company's test kit); The sequence of α-actin-IGF1 expression cassette shown in SEQ ID No.1,
Cut transposon carrier pT2-HB(that both sides contain forward and reverse transposable element by Hackett professor (Unibersity of Minnesota with the EcoRV enzyme simultaneously, USA) be so kind as to give, Zongbin Cui, Aron M.Geurts, Geyi Liu, Christopher D.Kaufman, Perry B.Hackett.StructureFunction Analysis of the Inverted Terminal Repeats of theSleeping Beauty Transposon.Journal of Molecular Biology, 318 (5): this carrier of 1221-1235. has detailed collection of illustrative plates and sequence information at Hackett professor's website, laboratory http://www.cbs.umn.edu/labs/perry/plasmids/plasmid.html), the purified product of α-actin-IGF1 expression cassette (containing IGF1 (C1:Ea)) is connected with carrier pT2-HB after enzyme is cut is built into pT2/ α-actin-IGF1 expression vector.PT2/ α-actin-IGF1 plasmid enzyme restriction qualification result as shown in Figure 6, pT2/ α-the actin-IGF1 plasmid map is as shown in Figure 7.
Table 1: the primer that is used for the amplification vector member among the present invention
Example II, the cell levels that contains the special sk-α of skeletal muscle-actin promoters driven GFP of the present invention are expressed check
1, the recovery of freeze-stored cell and cultivation
PEGFP-N1 plasmid (be PT3027-5 available from the Clontech article No.), pT2/ α-actin-GFP plasmid reclaim with QIAGEN purification kit glue, and the product final concentration is adjusted into 500ng/ul and is used for cell transfecting.
From liquid nitrogen, take out voluntarily separation and Culture of porcine fetus fibroblasts PEF(Institute of Animal Sciences, Chinese Academy of Agricultural Sciences cell engineering laboratory be housed), the frozen tubule of mouse C2C12 cell and pig PK15 cell (available from Chinese Academy of Sciences's cell bank), drop into immediately in 37-40 ℃ the warm water and rock fast, until frozen storing liquid melts fully; In 1-2min, finish rewarming; Cell suspension is moved into aseptic centrifuge tube, add the 5mL nutrient solution, blow gently even; With the centrifugal 5min of cell suspension 800-1000rpm, abandon supernatant; Add the 1mL perfect medium to the centrifuge tube that contains cell precipitation, blow gently evenly, change cell suspension over to Tissue Culture Flask, add an amount of perfect medium and cultivate.
2, cell transfecting and screening
Porcine fetus fibroblasts PEF, mouse muscle-forming cell C2C12 clone and porcine kidney cell PK15 clone are divided into respectively three groups (the blank pT2-HB plasmid of transfection control group, transfection pEGFP-N1 plasmid positive controls, transfection pT2/ α-actin-GFP plasmid group), every group of 2 repetitions.
24h before the transfection, contain to every hole 10% foetal calf serum (available from GIBCO company, article No.: 500 μ LDMEM high glucose mediums 21640-079) (available from GIBCO company, article No.: press 0.5-2 * 10 in 24 orifice plates 12100-046)
5Individual cells/well is inoculated respectively three kinds of clones, makes cell reach about 90% converge before transfection; Dilute respectively 0.8 μ g with the substratum of the serum-free of 50 μ L, antibiotic-free and treat transfection DNA, and gentle mixing; Before using the liposome gentleness is shaken up, with 2 μ Llipefectamin
TMThe DMEM substratum of-2000 serum-frees that add 50 μ L, antibiotic-free and mixing gently are in incubated at room 5min; Behind the 5min, respectively 50 μ L are respectively organized the liposome diluent that the DNA diluent adds 50 μ L, mixing and place 20min in room temperature gently; The DNA liposome mixture of 100 μ L is joined in the ready hole, rock back and forth gently culture plate, be placed on 37 ℃, saturated humidity, 5%CO
2Incubator in cultivate, behind 4h, replace transfection media with perfect medium, cultivate the transient expression intensity of under the 495nm wavelength, observing green fluorescence behind the 24-36h fully.
3, transfectional cell green fluorescence expression intensity is identified
The positive control group of reporter gene expression carrier pEGFP-N1 of the CMV promotor of expressing with wide spectrum, the pT2-HB plasmid is the blank group, pT2/ α-actin-GFP is experimental group, respectively transfection (a) sarcoplast C2C12, (b) porcine fetus fibroblasts PEF and (c) porcine kidney cell PK15, behind the transfection 24h, fluorescence microscopy Microscopic observation fluorescence.Found that: equal redgreen luciferase expression in take the pT2-HB plasmid as three kinds of clones of blank group, the fluorescence intensity among the sarcoplast C2C12 of pT2/ α-actin-GFP transfection is apparently higher than control group; And in porcine fetus fibroblasts PEF, the fluorescence intensity of pT2/ α-actin-GFP transfection group then is starkly lower than control group; In well differentiated porcine kidney cell PK15, pT2/ α-actin-GFP transfection group does not have the expression of fluorescence fully.(as shown in Figure 8).The result shows that the expression vector that makes up in the experiment has higher cell type specificity, confirmed in this experiment clone's promotor can mediate foreign gene the in vivo expression of differential high efficient in the muscle.
EXAMPLE III, contain the checking of the transgenic mice of pig IGF1 expression vector preparation
1, expresses the preparation of pig IGF1 transgenic mice
Ring-type pT2/ α-actin-IGF1 plasmid and the plasmid pCMV-SB11 that expresses transposase are mixed by the volumetric molar concentration such as 1: 1, utilize the pronuclear microinjection legal system for transgenic mice.
2, the integration of transgenic mice detects
Utilize respectively for sk-α-actin Promoter (P 1), sk-α-actin 3 ' UTR (P2) and pIGF1 total length (P3) and detect the integration that primer (seeing Table 2) detects respective carrier element in the transgenic mice genomic dna.
Detect the integration of foreign gene among the transgenic mice offspring who obtains in 1.The PCR reaction system is 20 μ l, 2 μ L10 * Buffer, 2 μ L 2.5mM dNTP, 1 μ L, 20 μ M primers F, 1 μ L, 20 μ M primer R, 0.5 μ L5 U/ μ L high-fidelity Tag polysaccharase, and 100ng mouse tail tissue DNA adds ultrapure water to 20 μ L.Pcr amplification program: 95 ° of C 5min; 94 ° of C 30s, 60 ℃ of 30s, 72 ° of C 1min circulate 30 times, and last 72 ° of C extend 5min.Pcr amplification product detects with 1.5% agarose gel electrophoresis, filtering out the mouse that can amplify respectively sk-α-actin Promoter, sk-α-actin 3 ' UTR and pIGF1 gene is that transgenic positive mouse (Fig. 9) is (among the figure, blank: negative control, normal mice DNA are template; 1-20: be part transgenic mouse amplified band, upper figure is visible 5,7, the 15-18 mouse can amplify the expection band).
In the table 2 as detecting the primer (the synthesising probing needle primer sees Table 2) of integrative vector element and label probe product, according to test kit process specifications system application of sample; The PCR working procedure is as follows: 95 ℃ of 5min, and 94 ℃ of 30S, 60 ℃ of 1min, 72 ℃ of 1min, rear 72 ℃ of 30 circulations are extended 5min(probe mark test kits available from Roche company, article No.: 11175033910).
Carry out the southen hybridization check take the PCR product as probe, before the hybridization 95 ℃ of sex change after 5 minutes immediately in the frozen water cooling for subsequent use; (Animal genome DNA extraction test kit is available from Roche company, article No.: 11814770001), cut genome with the EcoRV enzyme, separate each endonuclease bamhi in 1% agarose gel electrophoresis, every hole loading 5ug for a large amount of extracting mouse tail tissue DNAs; Then transfer on the positively charged nylon membrane after making the sex change of DNA original position.According to the test kit operation instructions carry out prehybridization, hybridization and X-mating plate exposure tests hybridization signal (available from Roche company, article No.: 11745832910).Detected result (Figure 10) shows that PCR detects positive mouse sample, the corresponding mouse of southern hybridization check can detect respective strap (among the figure ,+: for containing the plasmid DNA of target gene, as positive control; The transgenic mouse of 1-9 sample representative is that target gene is integrated mouse;-: for the genomic dna of wild-type mice, as negative control).
3, the preparation of the total RNA extraction of mouse tissue and cDNA
The positive transgenic mice that sifts out from 2 is normal raised to 16 ages in week, butcher to get each histoorgan and be used for extracting RNA, according to sky root total RNA from animal tissues extract test kit (article No.: DP431) operation instructions, extract the total RNA of each histocyte; According to Ferment reverse transcription test kit (article No.: K0441), total RNA reverse transcription of extracting is become cDNA.
4, IGF1 transgenic mice fluorescence quantitative PCR detection
The cDNA of preparation is as template in 3, respectively take IGF1 and mouse β-actin gene as confidential reference items (the step 1(primer of amplification method such as example I sees Table 2)), carry out quantitative fluorescent PCR and measure the expression amount of IGF1, the loading system is according to ABI quantitative reagent specification sheets loading, reaction system is 20 μ l, 10 μ L, 2 * Mixturer (available from TaKaRa company, article No. DRR041A), 1 μ L, 20 μ M upstream primers, 1 μ L, 20 μ M downstream primers, 1 μ L cDNA add ultrapure water to 20 μ L.The pcr amplification program: 95 ° of C 30s, 95 ° of C 5s, 60 ℃ of 34s circulate 40 times, and last 72 ° of C extend 5min.
The fluorescent quantitation detected result as shown in figure 11, when carrying out the detection of IGF1 expression level, take wild-type mice (control group) liver organization expression amount as benchmark, the expression amount of IGF1 is 15 times of control group in the transgenic mice skeletal muscle, and the expression amount in the expression amount of IGF1 and the control group respective organization is suitable in its hetero-organization, confirms that trans-exogenous IGF1 expression cassette can differential high efficient ground overexpression in skeletal muscle.
Table 2 transgenic mouse Screening and Identification and quantitative primer
5, IGF1 transgenic mice Western blot detects
(total protein extraction agent box is available from Thermo company to extract total protein in the positive transgenic mice sample that sifts out from 2 and 4, article No.: 78510), after protein electrophoresis, transferring film, (anti-human IGF1 antibody is available from Abcam company to carry out hybridization check with anti-human IGF1 primary antibodie, article No.: ab106836), and carry out conventional compressing tablet, exposure tests hybridization signal.
Western blot detects the protein expression result as shown in Figure 12 and Figure 13, and external source IGF1 protein expression profiles is presented at high expression level in the transgenic mice skeletal muscle, simultaneously trace expression is arranged also in cardiac muscle, and nothing is expressed in its hetero-organization; IGF1 expresses and to compare with the expression level of IGF1 in the normal mouse skeletal muscle in the transgenic mice skeletal muscle, apparently higher than normal mouse, gray-scale value is quantized relatively, finds that IGF1 expressing quantity in the transgenic mice skeletal muscle is about 3.5 times of normal mouse.Confirmed that transgene expression vector can promote external source IGF1 overexpression specifically in skeletal muscle in vivo.
Claims (3)
1. the expression cassette of the special sk-α of a boar endogenous bone bone flesh-actin promoters driven IGF1 gene is comprised of the sk-α that connects successively-actin promotor, pig IGF1 gene, sk-α-actin 3 ' UTR and SV40 enhanser; Its sequence is shown in SEQ ID No.1.
2. the special expression pig IGF1 genetic safety type expression vector of crossing of skeletal muscle is characterized in that containing the special sk-α of pig endogenous bone flesh claimed in claim 1-actin promoters driven expression cassette; The sequence of this expression vector is shown in SEQ ID No.2.
3. carrier according to claim 2 is characterized in that: be that the special sk-α of the described pig endogenous of claim 1 bone flesh-actin promoters driven expression cassette is cloned among the transposon carrier pT2-HB by the EcoRV restriction enzyme site and is obtained.
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Application publication date: 20130320 |