CN101984062B - Transcriptional control DNA in vitro transcription - Google Patents
Transcriptional control DNA in vitro transcription Download PDFInfo
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- CN101984062B CN101984062B CN2010101824049A CN201010182404A CN101984062B CN 101984062 B CN101984062 B CN 101984062B CN 2010101824049 A CN2010101824049 A CN 2010101824049A CN 201010182404 A CN201010182404 A CN 201010182404A CN 101984062 B CN101984062 B CN 101984062B
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Abstract
The invention relates to the establishment of vitro transcription plasmids of 6 pig source key genes for maintaining the self-renewal of stem cells, which is characterized by comprising the following steps: firstly designing a vitro transcription null vector Vitro-Trans PMD-18T of the genes; obtaining enhanced green fluorescent proteins (EGFP) and complementary Deoxyribose Nucleic Acid (cDNA) of the 6 pug source key genes for maintaining the self-renewal of the stem cells and carrying out the enzyme cutting connection according to the corresponding enzyme cutting sites; carrying out the vitro transcription of 7 vitro transcription plasmids by utilizing a vitro transcription kit to obtain the corresponding messenger Ribose Nucleic Acid (mRNA); transfecting Vitro-Trans-EGFP into an embryo of a pig to form fiber cells; and transfecting the mRNA from the vitro transcription of the 6 genes into the embryo of the pig to be converted into pluripotent stem cells of the pig after forming the fiber cells, wherein the mRNA from the vitro transcription of the 6 genes can be translated at high efficiency.
Description
Technical field:
The present invention utilizes genetic engineering technique; Make up the in-vitro transcription carrier and can induce the pig embryo fibroblast to be transformed into the in-vitro transcription plasmid of 6 key genes of pig multipotential stem cell; Be used to induce the inoblast of pig to become the pig multipotential stem cell; For setting up the human diseases model pacing items is provided, this invention belongs to the genetically engineered field.
Background technology:
Pig is best suited for the animal that the human organ transplants and sets up the human diseases model; But the disease model of pig great majority utilize the technology of nuclear transplantation to produce the ovocyte that can continue to grow with the somatocyte that the gene knockout technology obtains gene knockout then, thereby obtain gene knock-out pig.But in this process, exist gene knockout efficient low, the pregnancy period abortion ratio is high, problems such as newborn piglet defectiveness; In addition, the stem cell of pig separates so far not success, so according to the experimental technique of Yamanaka research group, the key gene that in inoblast, changes several stem cells over to just can be transformed into multipotential stem cell with inoblast.At present; Utilize transfection reagents such as liposome very low to fibroblastic multi-gene transfected efficient; The gene dosage that causes changing over to is different, infects the problem that there is biological safety again in gene knock-out pig that fibroblastic method obtains but carry gene with retrovirus with it.Therefore, we attempt 6 key genes of transcribing the pig of keeping the stem cell self in a large number external, make it become the multipotential stem cell of pig after changing its mRNA over to the pig embryo fibroblast then.
Summary of the invention:
One of the object of the invention is to make up the outer transcription vector of genosome, and this carrier can carry out a large amount of in-vitro transcription to certain gene, obtains the mRNA that can efficiently translate.
Another object of the present invention is to have made up 6 key genes carrying the pig of keeping the stem cell self and the in-vitro transcription plasmid of crt gene EGFP, and the mRNA transfection that these 6 gene in-vitro transcription go out can make it be transformed into the multipotential stem cell of pig after advancing the pig embryo fibroblast; And identified the function of in-vitro transcription mRNA through cell transfecting with cell levels.
Technical scheme of the present invention is achieved in that the structure of the in-vitro transcription plasmid of 6 pig source key genes keeping the stem cell self, it is characterized in that concrete preparation method is following:
At first design gene in-vitro transcription empty carrier Vitro-TransPMD-18T; Obtain the cDNA of EGFP and 6 key genes in the pig source of keeping the stem cell self then; Carry out enzyme by corresponding restriction enzyme site again and cut connection, and 7 external plasmids of transcribing are carried out in-vitro transcription, obtain corresponding mRNA with the in-vitro transcription test kit; Then the pig embryo fibroblast is advanced in the Vitro-Trans-EGFP transfection, detect its function;
1, the structure of the outer transcription vector of genosome
Obtain the sequence of 104bp with the method for overlapping extension PCR, comprise the T7 promotor, strengthen the minusCT of translation efficiency, comprise 5 ' UTR and one section irrelevant sequence that comprises restriction enzyme site of ATG initiator codon.Connect then and get into PMD-18Tsimple carrier, the insertion of expressing gene after being used for.
According to 2 pairs of primers of aim sequence design, P1, P2; P3, P4.Carry out the sequence that pcr amplification obtains 60bp with primer P1 and P2, electrophoresis is cut glue and is reclaimed, and the product with 60bp is that template is carried out PCR with primer P3 and P4 then, obtains the reverse transcription template sequence of 104bp.
Table 1 primer sequence
| Primer | Nucleotide sequence |
| P1 | 5′-tatagggagaagagggagagtagagccgccaccat?3′ |
| P2 | 5′-attctccgcggacatatgtgggcccatggtggcgg3′ |
| P3 | 5′-Ctcgaggactaatacgactcactataggga?3′ |
| P4 | 5′-aagcttgctagctctcgagagaattctccg?3′ |
Carry out being connected of in-vitro transcription template sequence and skeleton carrier according to the specification sheets of PMD-18T simple carrier.Proved the correct structure of in-vitro transcription carrier then with the enzyme blanking method.
2, keep the acquisition of cDNA of the pig source key gene of stem cell self
Utilize the sequence of the EGFP that announces among the NCBI and the corresponding gene mRNA sequence of duroc; The design primer; Comprise: Oct4 (NM_001113060.1), Sox2 (NM_001123197.1), c-Myc (NM_001005154.1); Klf4 (NM_001031782.1), Nanog (NM_001129971.1) and Lin28 (NM_001123133.1); The primer two ends have the restriction enzyme site (NcoI, EcoRI and NheI) of the outer transcription vector of linker, and the method for employing RT-PCR obtains the cDNA sequence of these 6 genes;
3, the acquisition of gene in-vitro transcription plasmid
With above-mentioned in-vitro transcription empty carrier and Oct4, Sox2, c-Myc, Klf4, the cDNA of 7 genes of Nanog and Lin28 is connected, and obtains gene in vitro and transcribes plasmid (VitroTrans-EGFP/Oct4/Sox2/c-Myc/Klf4/Nanog/Lin28);
4, gene in vitro is transcribed
Utilize the mMESSGE mMACHINE T7Ultra Kit of Applied Biosystems company that linearizing gene in-vitro transcription plasmid is carried out in-vitro transcription;
5, the checking of the mRNA function that goes out of gene in-vitro transcription
The mRNA that is used as the EGFP that the in-vitro transcription of contrast goes out carries out transfection to the pig inoblast, observes egfp expression after 12 hours.
The sequence of the outer transcription vector of described genosome and contain the carrier of similar elements identical function.
The detection---fluoroscopic examination of Test Example 1Vitro-Trans-EGFP mRNA transfection pig embryo fibroblast
The EGFP mRNA transient transfection China landrace embryo fibroblast that 1 pig embryo fibroblast obtains in-vitro transcription.
2 processs of the test inoculation 1 * 10
5In individual cell to 6 orifice plate, be with its density to reach to about 80% and carry out transfection, transfection is with the lipofectamine LTX+plus of invitrogen company.DMEM with 10%FBS before the transfection changes Opti-MEM2 hour into, transfection 2ug EGFP mRNA, and transfection after 6 hours changes Opti-MEM into the DMEM of 10%FBS.37 degree, 5%CO
2After cultivating 12 and 24 hours respectively, observe the expression of green fluorescent protein at the fluorescence co-focusing microscopically.
The pig embryo fibroblast expressing green fluorescent protein of 3 test-results transfection EGFP mRNA, expression time can continue 72 hours.Be beneficial to it and carry out inducing of multipotential stem cell.The result sees Fig. 1:
Positively effect of the present invention is can make it be transformed into the multipotential stem cell of pig after the pig embryo fibroblast is advanced in mRNA transfection that 6 gene in-vitro transcription go out; The mRNA-that 6 gene in-vitro transcription go out can efficiently translate.
Description of drawings:
Figure 1A .EGFP mRNA transfection egfp expression situation after 12 hours.
Figure 1B. the white light source cell density situation of Figure 1A.
Fig. 1 C.EGFP mRNA transfection egfp expression situation after 24 hours.
The white light source cell density situation of Fig. 1 D. Fig. 1 C.
Fig. 2 A. in-vitro transcription carrier figure
Fig. 2 B. in-vitro transcription carrier sequence
Embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is done further description:
The structure of the outer transcription vector of embodiment 1 genosome
Confirming of 1 sequence: be transcribed into the condition of the mRNA that can efficiently translate outward according to genosome, we select the T7 promotor to start to transcribe; In addition, the CT sequence that we design 14bp according to document and many years of experience is transcribed efficient in order to enhancing; We have designed sequence the synthesizing in order to startup mRNA that comprises the ATG initiation site at the restriction enzyme site place that inserts gene at last.Sequence is seen Fig. 2: Fig. 2 A. in-vitro transcription carrier figure; Fig. 2 B. in-vitro transcription carrier sequence 2 design of primers, overlapping extension PCR reach and are connected with the T carrier: according to designed sequence, we design 2 pairs of overlapping primers, and wherein the overlapping 10bp of P1 and P2 respectively contains 35 bases.P3 and P4 respectively with overlapping 7 bases of the product of P1 and P2, respectively contain 30 bases.The annealing temperature of P1 and the overlapping extension of P2 primer is 65 degree, is primer with P3 and P4, and the PCR product of P1 and P2 is that the annealing temperature of template also is 65 degree.Final PCR product is 104bp.Then the 104bp sequence that obtains is connected with PMD-18T, subsequent use.
Embodiment 2 crt gene EGFP and the acquisition of keeping 6 key gene cDNA of stem cell self
1 sequence is confirmed and design of primers: the Oct4 of inquiry EGFP and pig in ncbi database, Sox2, c-Myc, Klf4, Nanog, Lin28mRNA sequence, the PCR primer of its sequence of design amplification then.Oct4 (NM_001113060.1), Sox2 (NM_001123197.1), c-Myc (NM_001005154.1), Klf4 (NM_001031782.1), Nanog (NM_001129971.1) and Lin28 (NM_001123133.1).According to sequence and the correct principle of optimality of expressing that inquiry obtains, design primer.The primer two ends have the restriction enzyme site (NcoI, EcoRI and NheI) of the outer transcription vector of linker.
2RT-PCR and with being connected of T carrier: in the farrowing sow body, go out the embryo about 20 days, liquid nitrogen grinding is carried out the extraction of RNA then; Carry out rt again; CDNA with rt is a template then, carries out the pcr amplification of 6 genes, thereby obtains the cDNA of specific 6 genes in pig source.Also have EGFP to be connected with the TOPO-PCR carrier cDNA product of each gene that reclaims, conversion is shaken bacterium and is obtained carrying out enzyme behind the corresponding plasmid and cut evaluation.
37 genes of embodiment are connected with the in-vitro transcription carrier
The enzyme of 17 genes and in-vitro transcription carrier is cut: the Oct4-T plasmid is cut with EcoRI and NheI enzyme, Sox2-T, and c-Myc-T, Klf4-T, Nanog-T and Line8-T plasmid are cut with NcoI and NheI enzyme.Electrophoresis is cut glue then, reclaims.The in-vitro transcription carrier also carries out enzyme with corresponding enzyme simultaneously and cuts.Endonuclease bamhi with 7 genes is connected with the in-vitro transcription carrier then, transforms, and shakes the dientification of bacteria.
27 gene in vitro transcription vectors linearizing: will identify that 7 correct gene in vitro transcribe plasmid and carry out enzyme with NheI and cut and make its linearizing, and be beneficial to the carrying out of in-vitro transcription.
47 gene in vitro of embodiment are transcribed
According to the testing sequence of mMESSAGE mMACHINE T7Ultra Kit, in the environment that no RNA enzyme pollutes, 7 linearizing genes are carried out in-vitro transcription, and accomplish the process that adds cap and add the PolyA tail.And identify.
The checking of the mRNA function that embodiment 5 gene in-vitro transcription go out
The mRNA transfection of the VitroTrans-EGFP that obtains is advanced in 35 days the embryo fibroblast of pig, observe its fluorescent protein expression situation after 12 and 24 hours.
Sequence table
Application?Project
------------------
< 120>Title: the structure of in-vitro transcription plasmid of keeping 6 pig source key genes of stem cell self
< 130>AppFileReference: files reference number
<160>1
<210>1
Sequence
--------
< 213>OrganismName: artificial sequence
<400>PreSequenceString：1
ctcgaggact?aatacgactc?actataggga?gaagagggag?agtagagccg?ccaccatggg 60
cccacatatg?tccgcggaga?attctctcga?gagctagcaa?gctt 104
<212>Type：DNA
<211>Length：104
SequenceName：Vitro-TransPMD-18T
Claims (2)
1. in-vitro transcription regulating DNA is characterized in that sequence is following:
Ctcgaggactaatacgactcactatagggagaagagggagagtagagccgccaccatgggcccacatatgtccgcggagaattctctcgagagctagcaagctt
2. the compound method of in-vitro transcription regulating DNA is characterized in that: comprise the T7 promotor, strengthen the minusCT of translation efficiency, comprise 5 ' UTR and one section irrelevant sequence that comprises restriction enzyme site of ATG initiator codon, be used for the insertion and the in-vitro transcription of goal gene; Method is overlapping extension PCR method, according to 2 pairs of primers of aim sequence design,
P1：5′-tatagggagaagagggagagtagagccgccaccat?3′
P2：5′-attctccgcggacatatgtgggcccatggtggcgg3′
P3：5′-ctcgaggactaatacgactcactataggga?3′
P4：5′-aagcttgctagctctcgagagaattctccg?3′
Carry out PCR with primer P1 and P2, amplification obtains the sequence of 60bp, and electrophoresis is cut glue and reclaimed, and the product with 60bp is that template is carried out PCR with primer P3 and P4 then, obtains the transcription regulating nucleotide sequence of 104bp.
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| CN2010101824049A CN101984062B (en) | 2010-05-26 | 2010-05-26 | Transcriptional control DNA in vitro transcription |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5547862A (en) * | 1993-07-29 | 1996-08-20 | Ambion Inc. | Vectors containing multiple promoters in the same orientation |
| US5691140A (en) * | 1995-05-18 | 1997-11-25 | New England Biolabs, Inc. | Bidirectional in vitro transcription vectors utilizing a single RNA polymerase for both directions |
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- 2010-05-26 CN CN2010101824049A patent/CN101984062B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5547862A (en) * | 1993-07-29 | 1996-08-20 | Ambion Inc. | Vectors containing multiple promoters in the same orientation |
| US5691140A (en) * | 1995-05-18 | 1997-11-25 | New England Biolabs, Inc. | Bidirectional in vitro transcription vectors utilizing a single RNA polymerase for both directions |
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