CN114752625A - Goat-derived OSKM as well as construction method and application thereof - Google Patents
Goat-derived OSKM as well as construction method and application thereof Download PDFInfo
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Abstract
The invention provides a goat-derived OSKM as well as a construction method and application thereof, belonging to the technical field of biomedicine. The method for constructing the goat source OSKM provided by the invention obtains Oct-4, Sox-2 and c-Myc genes by PCR amplification by taking goat embryo cDNA as a template; PCR amplification is carried out by taking goat fetal ear fibroblast cDNA as a template to obtain Klf-4 gene, and the four genes are respectively provided with 2A peptide sequences to obtain four gene fragments; and connecting the four gene fragments with a linearized vector after the four gene fragments are respectively provided with 15-20bp recognition sites to obtain the goat-derived OSKM. The goat-derived OSKM constructed by the method can successfully induce the reprogramming of goat fibroblasts, and has high reprogramming efficiency.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a goat-derived OSKM as well as a construction method and application thereof.
Background
The embryonic stem cell has two characteristics of self-renewal capacity and differentiation pluripotency, so that the possibility of obtaining various cells, tissues and even organs in vitro is realized. However, human embryonic stem cells are derived from human embryos and have ethical problems; in addition, when the embryonic stem cells are really applied from other individuals, the problem of immunological rejection cannot be solved, so that the embryonic stem cells are greatly limited in practical clinical application. And the Induced Pluripotent Stem Cells (iPSC) obtained by reprogramming the somatic cells can effectively avoid the ethical problem and the immunological rejection problem of the embryonic stem cells in the application process.
At present, the method for inducing the reprogramming of the pluripotent stem cells of the livestock in the field mostly takes retrovirus vectors, lentivirus vectors, adenovirus vectors, plasmid vectors and the like as common vectors for preparing iPSC. However, retroviral vectors and lentiviral vectors cause mutations when a carrier gene is inserted into a host, and may change the expression pattern of adjacent genes, which has the disadvantage of increasing the risk of causing tumors in iPSC cells, and the induction efficiency of adenoviral vectors and plasmid vectors is very low. Although researches prove that mouse and human adult cells can be reprogrammed to be pluripotent stem cells by utilizing a polyprotein expression system and chemical small molecules and synthesizing mRNA (messenger ribonucleic acid), the methods avoid the possibility of inserting exogenous genes into somatic cell chromosomes, but the operation process of inducing iPSC (induced pluripotent stem cells) is complicated, and the efficiency is extremely low.
Induction of the reprogramming process of pluripotent stem cells requires the delivery of transcription factors associated with the proliferation, renewal and pluripotency of Embryonic Stem Cells (ESCs) to somatic cells, which in turn triggers the transcriptional and translational activation of endogenous pluripotency factors, inducing their conversion into ESCs-like cells. The genes commonly used in the studies to establish livestock ipscs are Takahashi's classical four-factor OSKM: oct-4, Sox-2, c-Myc and Klf-4, because the construction of autologous OSKM gene is easy to cause Klf-4 gene mutation, and further the in vitro reprogramming can not be achieved, the OSKM gene used in the goat somatic cell reprogramming report is all other species. Due to species intergenic differences, the use of other species seed genes will affect reprogramming efficiency. In addition, although a host gene is easily mutated when a viral vector is inserted into a host, the transfection efficiency of the viral vector is higher than that of a plasmid vector, and thus viral vectors are often used in the field of in vitro reprogramming using the OSKM four-factor. Therefore, how to construct and obtain an autologous OSKM which can be successfully used for in vitro reprogramming and how to improve the transfection efficiency of in vitro reprogramming by using a plasmid vector are technical problems to be solved urgently in the field.
Disclosure of Invention
In view of the above, the invention aims to provide a method for constructing goat Oct-4, Sox-2, c-Myc and Klf-4 gene expression vectors, and the goat OSKM constructed by the method can significantly improve the efficiency of reprogramming goat somatic cells.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for constructing goat OSKM, which comprises the following steps: performing PCR amplification by taking goat somatic cells as templates to obtain Oct-4, Sox-2, c-Myc and Klf-4 genes, and connecting the Oct-4, Sox-2, c-Myc and Klf-4 genes with a linearized vector to obtain a goat-derived OSKM; the primer sequences of Oct-4, Sox-2, c-Myc and Klf-4 genes obtained by amplification are as follows:
Oct-4：F:ATGGCGGGACACC，R:TCAGTTTGCATGC；
Sox-2：F:ATGTACAACATGA，R:TCACATGTGCGAG；
c-Myc：F:ATGCCCCTCAACG，R:TTAGGCGCAAGAG；
Klf-4：F:ATGAGGCAGCCAC，R:TTAAAAGTGCCTT。
preferably, the Oct-4, Sox-2, c-Myc and Klf-4 genes are obtained by PCR amplification at annealing temperatures of 54 deg.C, 56 deg.C, 60 deg.C and 64 deg.C, respectively.
Preferably, the connecting comprises the following steps: taking Oct-4, Sox-2, c-Myc and Klf-4 gene fragments as templates, carrying out PCR amplification to ensure that the Oct-4, Sox-2, c-Myc and Klf-4 genes respectively have 2A peptide sequences to obtain four gene fragments; four gene fragments are taken as templates, PCR amplification is carried out, the four gene fragments are respectively provided with 15-20bp recognition sites, and then the four gene fragments are connected with a linearized vector.
Preferably, the PCR primers for allowing the Oct-4, Sox-2, c-Myc and Klf-4 genes to have 2A peptide sequences are:
Oct-4+2A：
F:ATGGCGGGACACCTCGCT
R:aatcaaagttaagagtttgtttgacaggagcgacaattttTCAGTTTGCATGCAT；
Sox-2+2A：
F:TACTCAAACTGGCTGGGGATGTAGAAAGCAATCCAGGTCCAATGTACAACATGATG
R:aatcaaagttaagagtttgtttgacaggagcgacaatttttttTCACATGTGCGAGAG；
c-Myc+2A：
F:TACTCAAACTGGCTGGGGATGTAGAAAGCAATCCAGGTCCAATGCCCCTCAACGTC
R:aatcaaagttaagagtttgtttgacaggagcgacaatttttttTTAGGCGCAAGAGTT
Klf-4+2A:
F:TACTCAAACTGGCTGGGGATGTAGAAAGCAATCCAGGTCCAATGGCTGTCAGCGAC
R:TTAAAAGTGCCTCTTCATGT。
preferably, the PCR primers for making each of the four gene fragments have 15-20bp recognition sites are:
Oct-4+clon：F:gatcggccggatatcgaattcATGGCGGGACACCTCGCT
R:ccccagccagtttgagtaAATCAAAGTTAAGAGTTTGTTTGACAGG；
Sox-2+clon：F:ttTACTCAAACTGGCTGGGGATG
R:ccccagccagtttgagtaAATCAAAGTTAAGAGTTTGTTTGACAGG；
c-Myc+clon：F:ttTACTCAAACTGGCTGGGGATG
R:cccagccagtttgagtaAAGATTTGGCTCAATTATATATTTGAAGG；
Klf-4+clon：F:cttTACTCAAACTGGCTGGGGATG
R:aggcgcgccaagcttgcggccgcTTAAAAGTGCCTCTTCATGTGTAAGG。
preferably, the linearized vector is: and (3) carrying out enzyme digestion on the plasmid PB-TRE-hRL, and recovering the PB-TRE skeleton of the vector.
The invention also provides the goat-derived OSKM constructed by the method.
The invention also provides an application of the method or the goat-derived OSKM in inducing goat fibroblast reprogramming.
The invention also provides a method for inducing the reprogramming of goat fibroblasts, which comprises the following steps: transferring the reprogramming carrier into goat fibroblasts, and culturing in a culture medium, wherein the reprogramming carrier comprises the goat-derived OSKM.
Preferably, the reprogramming vector further comprises: pBase, EF1 alpha, pNhL, hRL and sLargeT plasmids.
The invention has the beneficial effects that:
the invention provides a method for constructing goat-derived OSKM for the first time, and the goat-derived OSKM constructed by the method can obviously improve the reprogramming efficiency of goat somatic cells.
Drawings
FIG. 1 shows the procedure of PCR amplification;
FIG. 2 is the results of the cloning culture on day 7 in the goat somatic cell reprogramming method of example 2;
FIG. 3 shows the morphology of goat fibroblasts clonally cultured without reprogramming;
FIG. 4 is the results of the clone culture on day 7 in the goat somatic cell reprogramming method of example 3.
Detailed Description
The invention provides a method for constructing goat OSKM, which comprises the following steps: performing PCR amplification by taking goat somatic cells as a template to obtain Oct-4, Sox-2, c-Myc and Klf-4 genes, and connecting the Oct-4, Sox-2, c-Myc and Klf-4 genes with a linearized vector to obtain a goat-derived OSKM; the primer sequences of Oct-4, Sox-2, c-Myc and Klf-4 genes obtained by amplification are as follows: oct-4: ATGGCGGGACACC (SEQ ID NO.1), R TCAGTTTGCATGC (SEQ ID NO. 2); sox-2: ATGTACAACATGA (SEQ ID NO.3), R TCACATGTGCGAG (SEQ ID NO. 4); c-Myc: ATGCCCCTCAACG (SEQ ID NO.5), R TTAGGCGCAAGAG (SEQ ID NO. 6); klf-4: ATGAGGCAGCCAC (SEQ ID NO.7) and R TTAAAAGTGCCTT (SEQ ID NO. 8).
In the present invention, the goat somatic cells preferably include goat embryo and goat fetus ear fibroblasts. In the specific embodiment of the invention, the Oct-4, Sox-2 and c-Myc genes are obtained by amplification with goat embryo cDNA as a template; and (3) amplifying by taking the goat fetal ear fibroblast cDNA as a template to obtain the Klf-4 gene. The present invention is not particularly limited in the reaction system for PCR amplification, and is not particularly limited in the denaturation temperature and extension temperature for PCR amplification, and when Oct-4, Sox-2, c-Myc and Klf-4 genes are obtained by PCR amplification using goat somatic cells as templates, the annealing temperatures are preferably 54 ℃, 56 ℃, 60 ℃ and 64 ℃, respectively.
In the present invention, the connection preferably includes the steps of: performing PCR amplification by taking Oct-4, Sox-2, c-Myc and Klf-4 gene fragments as templates to enable Oct-4, Sox-2, c-Myc and Klf-4 genes to respectively carry 2A peptide sequences to obtain four gene fragments; and performing PCR amplification by taking the four gene fragments as templates to ensure that the four gene fragments are respectively provided with 15-20bp recognition sites, and then connecting the four gene fragments with a linearized vector.
In the present invention, PCR primers for allowing Oct-4, Sox-2, c-Myc and Klf-4 genes to have 2A peptide sequences are preferably: oct-4+ 2A: ATGGCGGGACACCTCGCT (SEQ ID NO.9), R aatcaaagttaagagtttgtttgacaggagcgacaattttTCAGTTTGCATGCAT (SEQ ID NO. 10);
Sox-2+2A：
F:TACTCAAACTGGCTGGGGATGTAGAAAGCAATCCAGGTCCAATGTACAACATGATG(SEQ ID NO.11)，
R:aatcaaagttaagagtttgtttgacaggagcgacaatttttttTCACATGTGCGAGAG(SEQ ID NO.12)；
c-Myc+2A：
F:TACTCAAACTGGCTGGGGATGTAGAAAGCAATCCAGGTCCAATGCCCCTCAACGTC(SEQ ID NO.13)
R:aatcaaagttaagagtttgtttgacaggagcgacaatttttttTTAGGCGCAAGAGTT(SEQ ID NO.14)
Klf-4+2A:
F:TACTCAAACTGGCTGGGGATGTAGAAAGCAATCCAGGTCCAATGGCTGTCAGCGAC(SEQ ID NO.15)
TTAAAAGTGCCTCTTCATGT (SEQ ID NO. 16). In the present invention, there is no particular limitation on the PCR amplification system in which the Oct-4, Sox-2, c-Myc and Klf-4 genes are each provided with a 2A peptide sequence, and there are no particular limitations on the denaturation temperature and the extension temperature of PCR amplification, and the annealing temperature is preferably 60 ℃.
In the present invention, the PCR primers for allowing each of the four gene fragments to have a 15-20bp recognition site are preferably:
Oct-4+clon：F:gatcggccggatatcgaattcATGGCGGGACACCTCGCT(SEQ ID NO.17)，
R:ccccagccagtttgagtaAATCAAAGTTAAGAGTTTGTTTGACAGG(SEQ ID NO.18)；
Sox-2+clon：F:ttTACTCAAACTGGCTGGGGATG(SEQ ID NO.19)
R:ccccagccagtttgagtaAATCAAAGTTAAGAGTTTGTTTGACAGG(SEQ ID NO.20)；
c-Myc+clon：F:ttTACTCAAACTGGCTGGGGATG(SEQ ID NO.21)
R:cccagccagtttgagtaAAGATTTGGCTCAATTATATATTTGAAGG(SEQ ID NO.22)；
Klf-4+clon：F:cttTACTCAAACTGGCTGGGGATG(SEQ ID NO.23)
aggcgcgccaagcttgcggccgcTTAAAAGTGCCTCTTCATGTGTAAGG (SEQ ID NO. 24). The present invention is not particularly limited to a PCR amplification system in which each of the four gene fragments has a recognition site of 15-20bp, and is not particularly limited to a denaturation temperature and an extension temperature for PCR amplification, and the annealing temperature is preferably 60 ℃.
In the present invention, the linearized vector is preferably: and (3) carrying out enzyme digestion on the plasmid PB-TRE-hRL, and recovering the PB-TRE skeleton of the vector. The PB-TRE-hRL plasmid is given to Zhaolixia of inner Mongolian Seikexing research institute, and the PB-TRE-hRL plasmid is provided with a Dox (tetracyclines) regulation and control system and can regulate and control the expression of exogenous genes at any time. The Dox regulation system can strictly regulate the expression of exogenous genes, minimize the leakage expression of exogenous genes without an inducer, and maintain the high sensitivity of the system to Dox induction. The invention has no special limitation on the specific method for enzyme digestion of the plasmid PB-TRE-hRL, and has no special limitation on the specific recovery method of the vector PB-TRE skeleton.
The invention also provides the goat-derived OSKM constructed by the method and application of the method or the goat-derived OSKM in inducing goat fibroblast reprogramming.
The invention also provides a method for inducing the reprogramming of goat fibroblasts, which comprises the following steps: transferring the reprogramming carrier into goat fibroblasts, and culturing in a culture medium, wherein the reprogramming carrier comprises the goat-derived OSKM.
In the present invention, the reprogramming vector preferably further comprises: pBase, EF1 alpha, pNhL, hRL and sLargeT plasmids. The five plasmids of pBase, EF1 alpha, pNhL, hRL and sLargeT selected by the invention all belong to a PiggyBac vector system, and the plasmid is constructed by the PiggyBac vector system for the first time to induce the somatic cell of the goat to reprogram. The conventional plasmid transfection can only realize the transient expression of exogenous genes, and the exogenous genes are continuously lost along with the division of host cells, and are particularly remarkable in rapidly dividing cells. The PiggyBac transposon vector and the auxiliary plasmid are transfected into the mammalian cell simultaneously, and the target gene can be stably integrated into the chromosome of the host cell under the action of the transposase by the transposon, so that the target gene carried on the transposon vector is permanently expressed in the host cell. In the invention, the pNhL plasmid contains two genes of pig-derived NANOG and LIN28, is obtained from Nemongol Securin research institute Zhaocai, the hRL plasmid contains two genes of human-derived RARG and LRH1, is obtained from Nemongol Securin research institute Zhaocai, the sLargeT plasmid contains simian virus-derived LargeT, and is obtained from Liumouning of Nemongol agricultural university.
The specific method for transferring the reprogramming vector into the goat fibroblast is not particularly limited, and in the specific embodiment of the invention, the reprogramming vector is transferred into the goat fibroblast by adopting an electrotransfection method. In the invention, the reprogramming carrier is transferred into goat fibroblasts and cultured by using a culture medium, the culture medium used on the 1 st to 7 th days of culture preferably comprises M15, DOX, bFGF and LIF, the concentration of the bFGF and LIF is preferably 12ng/mL and 1000IU/mL respectively, the culture medium used on the 8 th to 14 th days of culture preferably comprises M15, DOX and LIF, and the concentration of the LIF is preferably 1000 IU/mL. The present invention is not particularly limited with respect to the specific sources of the components in the medium.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Construction of goat-derived OSKM
(1) Acquisition of goat-derived Oct-4, Sox-2, c-Myc and Klf-4 genes
Performing PCR amplification by taking goat embryo cDNA as a template to obtain Oct-4, Sox-2 and c-Myc genes; PCR amplification is carried out by taking goat fetal ear fibroblast cDNA as a template to obtain the Klf-4 gene. The primer sequences used for obtaining Oct-4, Sox-2, c-Myc and Klf-4 genes through PCR amplification are shown in Table 1, the system of PCR amplification is shown in Table 2, and the procedure of PCR amplification is shown in FIG. 1, wherein the annealing temperatures of Oct-4, Sox-2, c-Myc and Klf-4 are 54 ℃, 56 ℃, 60 ℃ and 64 ℃ respectively.
TABLE 1 amplification of primer sequences for Oct-4, Sox-2, c-Myc and Klf-4 genes
TABLE 2 PCR amplification System
(2) Obtaining linearized vectors
Plasmid PB-TRE-hRL (obtained from Zhaoli Xixia, Seikacin research institute of Nemontage) was digested with EcoR I and Not I, and the vector PB-TRE backbone was recovered after digestion, the digestion system is shown in Table 3. The enzyme digestion reaction conditions were 37 ℃ for 45 min.
TABLE 3 enzyme digestion System
Preparing 1.0% agarose gel for nucleic acid electrophoresis, and under the condition of 110V voltage, after the target band moves to a proper position, using TAKARA gel recovery kit to recover the linear vector gene fragment.
(3) Ligation of cloning vectors
Taking Oct-4, Sox-2, c-Myc and Klf-4 gene fragments obtained in the step (1) as templates, designing primers (shown in Table 4) to perform PCR amplification (a PCR amplification system is shown in Table 2, and the PCR amplification program is shown in figure 1, wherein the annealing temperature is 60 ℃) so that 2A peptide sequences are arranged between every two Oct-4, Sox-2, c-Myc and Klf-4 genes to obtain a gene fragment Oct-4-2A, Sox-2-2A, c-Myc-2A, Klf-4-2A;
TABLE 4 primer sequences for Oct-4, Sox-2, c-Myc, Klf-4 genes with 2A peptide sequences
Using Oct-4-2A, Sox-2-2A, c-Myc-2A, Klf-4-2A gene fragment as a template, designing primers (see Table 5) to perform PCR amplification (a PCR amplification system is shown in Table 2, and a PCR amplification program is shown in figure 1, wherein the annealing temperature is 60 ℃), so that each gene fragment of Oct-4-2A, Sox-2-2A, c-Myc-2A and Klf-4-2A is provided with a recognition site of 15-20bp, and gene fragments Oct-4-clone, Sox-2-clone, c-Myc-clone and Klf-4-clone are obtained;
TABLE 5 primer sequences for four gene fragments with 15-20bp recognition sites
The Vazyme Co., Ltd
The product of MultiS One Step Cloning Kit was used to ligate Cloning vectors, the ligation system is shown in Table 6.
TABLE 6 cloning vector ligation System
The prepared system is placed in a PCR instrument, reacted for 15min at 37 ℃, and then placed on ice for subsequent transformation experiments.
Plasmid amplification is carried out by using DH5a competence and then the obtained product is sent to Huada gene company for sequencing, and bacterium liquid with correct sequence is subjected to plasmid extraction by using a product of Plasmid Midi Kit of QIAGEN company, so that goat-derived OSKM is obtained.
Example 2
Somatic reprogramming in goats
The milk goat cell lines used in this experiment were all from the institute of Enmongolica Seisakusho.
Preparing feeder layer cells: the feeder cells were thawed to 10cm dishes 3-4 days before electrotransfection and cultured in M10 culture medium. The M10 medium was changed to M15+ DOX medium 1h before electrotransfection.
Preheating the required reagent at 37 ℃, placing the electrotransformation chamber at a temperature, and starting the electrotransformation instrument for preheating.
DNAmix was prepared from goat derived oskm (gsoskm) obtained in example 1 and plasmids pBase (obtained from inner mongolia seike institute of saikoxia), EF1 α (obtained from inner mongolia seike institute of saikoza of hutchxia), pNhL (obtained from inner mongolia seike institute of hutchxia), hRL (obtained from inner mongolia seike institute of hutchxia) and srarget (obtained from inner mongolia university of agriculture liumoning) as shown in table 7.
TABLE 7 DNA mix System
And discarding the fibroblast liquid and cleaning the DPBS. Adding 4mL of Trypsin-EDTA, and digesting for 3-5 min at 37 ℃. Digestion was stopped by adding 4mL of R and cells were resuspended. And (4) counting the cells. Will be 1 × 106The cells were transferred into a 15mL centrifuge tube, centrifuged at 1300r/min for 3min, and the solution was discarded. Add 100. mu.L of electrotransfer solution to the mixed DNA, mix well, add to the cell for resuspension, pipette to the electrotransfer cup. The electric rotor was placed in an electric rotor machine, and the procedure (U-023) was selected for electrotransfection. The electric beaker was removed, 1mL of preheated M15 medium was added, and the entire volume of the electric beaker was transferred to a 15mL centrifuge tube. The cells were electroporated as 1: 4 into a 10cm dish with feeder cells, and placed in a 38.5 ℃ incubator. The liquid was changed after 24h, and then every other day. The clone morphology can be observed in 5-7 days. The medium used 7 days after reprogramming was M15+ DOX + bFGF (12ng/mL) + LIF (1000IU/mL), and M15+ DOX + LIF (1000IU/mL) was used 7-14 days.
The compositions of the M10 culture medium and M15 culture medium used above are shown in Table 8 and Table 9, respectively.
The results of the 7 th day of the colony culture using the above-described goat somatic cell reprogramming method are shown in FIG. 2. The goat fibroblast cell which is not subjected to clone culture by the reprogramming method (see figure 3) is used as a control, so that the goat fibroblast reprogramming method can clone to obtain the iPSC within 7 days and has good cloning form. The invention uses 1X 10 in a 10cm cell culture dish 6Reprogramming the fibroblast cells, and finally725 clones are successfully obtained, and the reprogramming efficiency of the method is high and is better than that of the existing reprogramming method in the field.
Example 3
The difference from example 2 is that the feeder layer cell preparation step is: and (3) thawing the milk goat fibroblasts 3-4 days before electrotransfection to a 10cm culture dish, culturing by using an M10 culture solution, and carrying out cell passage for 4 generations. And performing electrotransfection when the fibroblast is converged to 60-70%. The M10 medium was changed to M15+ DOX medium 1h before electrotransfection. The cells were electroporated as 1: 8 into a 10cm dish containing feeder cells, and placed in a 38.5 ℃ incubator, the same as in example 2. The results of the colony culture on day 7 are shown in FIG. 4.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> university of inner Mongolia
INNER MONGOLIA SAIKEXING LIVESTOCK BREEDING AND SEED INDUSTRY BIOTECHNOLOGY RESEARCH INSTITUTE Co.,Ltd.
<120> goat source OSKM and construction method and application thereof
<160> 24
<170> SIPOSequenceListing 1.0
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<213> Artificial Sequence (Artificial Sequence)
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atggcgggac acc 13
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tcagtttgca tgc 13
<210> 3
<211> 13
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atgtacaaca tga 13
<210> 4
<211> 13
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tcacatgtgc gag 13
<210> 5
<211> 13
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
atgcccctca acg 13
<210> 6
<211> 13
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ttaggcgcaa gag 13
<210> 7
<211> 13
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
atgaggcagc cac 13
<210> 8
<211> 13
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ttaaaagtgc ctt 13
<210> 9
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
atggcgggac acctcgct 18
<210> 10
<211> 55
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
aatcaaagtt aagagtttgt ttgacaggag cgacaatttt tcagtttgca tgcat 55
<210> 11
<211> 56
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
tactcaaact ggctggggat gtagaaagca atccaggtcc aatgtacaac atgatg 56
<210> 12
<211> 58
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
aatcaaagtt aagagtttgt ttgacaggag cgacaatttt ttttcacatg tgcgagag 58
<210> 13
<211> 56
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
tactcaaact ggctggggat gtagaaagca atccaggtcc aatgcccctc aacgtc 56
<210> 14
<211> 58
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
aatcaaagtt aagagtttgt ttgacaggag cgacaatttt tttttaggcg caagagtt 58
<210> 15
<211> 56
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
tactcaaact ggctggggat gtagaaagca atccaggtcc aatggctgtc agcgac 56
<210> 16
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
ttaaaagtgc ctcttcatgt 20
<210> 17
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
gatcggccgg atatcgaatt catggcggga cacctcgct 39
<210> 18
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
ccccagccag tttgagtaaa tcaaagttaa gagtttgttt gacagg 46
<210> 19
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
tttactcaaa ctggctgggg atg 23
<210> 20
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
ccccagccag tttgagtaaa tcaaagttaa gagtttgttt gacagg 46
<210> 21
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
tttactcaaa ctggctgggg atg 23
<210> 22
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
cccagccagt ttgagtaaag atttggctca attatatatt tgaagg 46
<210> 23
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
ctttactcaa actggctggg gatg 24
<210> 24
<211> 49
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
aggcgcgcca agcttgcggc cgcttaaaag tgcctcttca tgtgtaagg 49
Claims (10)
1. A method for constructing goat-derived OSKM is characterized by comprising the following steps: performing PCR amplification by taking goat somatic cells as a template to obtain Oct-4, Sox-2, c-Myc and Klf-4 genes, and connecting the Oct-4, Sox-2, c-Myc and Klf-4 genes with a linearized vector to obtain a goat-derived OSKM; the primer sequences of Oct-4, Sox-2, c-Myc and Klf-4 genes obtained by amplification are as follows:
Oct-4：F:ATGGCGGGACACC，R:TCAGTTTGCATGC；
Sox-2：F:ATGTACAACATGA，R:TCACATGTGCGAG；
c-Myc：F:ATGCCCCTCAACG，R:TTAGGCGCAAGAG；
Klf-4：F:ATGAGGCAGCCAC，R:TTAAAAGTGCCTT。
2. The method according to claim 1, wherein the Oct-4, Sox-2, c-Myc and Klf-4 genes are obtained by PCR amplification at temperatures of 54 ℃, 56 ℃, 60 ℃ and 64 ℃, respectively.
3. The method according to claim 1, characterized in that said connecting comprises the steps of: performing PCR amplification by taking Oct-4, Sox-2, c-Myc and Klf-4 gene fragments as templates to enable Oct-4, Sox-2, c-Myc and Klf-4 genes to respectively carry 2A peptide sequences to obtain four gene fragments; four gene fragments are taken as templates, PCR amplification is carried out, the four gene fragments are respectively provided with 15-20bp recognition sites, and then the four gene fragments are connected with a linearized vector.
4. The method according to claim 3, wherein the PCR primers for allowing each of the Oct-4, Sox-2, c-Myc and Klf-4 genes to have a 2A peptide sequence are:
Oct-4+2A：
F:ATGGCGGGACACCTCGCT
R:aatcaaagttaagagtttgtttgacaggagcgacaattttTCAGTTTGCATGCAT；
Sox-2+2A：
F:TACTCAAACTGGCTGGGGATGTAGAAAGCAATCCAGGTCCAATGTACAACATGATG
R:aatcaaagttaagagtttgtttgacaggagcgacaatttttttTCACATGTGCGAGAG；
c-Myc+2A：
F:TACTCAAACTGGCTGGGGATGTAGAAAGCAATCCAGGTCCAATGCCCCTCAACGTC
R:aatcaaagttaagagtttgtttgacaggagcgacaatttttttTTAGGCGCAAGAGTT
Klf-4+2A:
F:TACTCAAACTGGCTGGGGATGTAGAAAGCAATCCAGGTCCAATGGCTGTCAGCGAC
R:TTAAAAGTGCCTCTTCATGT。
5. the method of claim 3, wherein the PCR primers for each of the four gene fragments with 15-20bp recognition sites are:
Oct-4+clon：F:gatcggccggatatcgaattcATGGCGGGACACCTCGCT
R:ccccagccagtttgagtaAATCAAAGTTAAGAGTTTGTTTGACAGG；
Sox-2+clon：F:ttTACTCAAACTGGCTGGGGATG
R:ccccagccagtttgagtaAATCAAAGTTAAGAGTTTGTTTGACAGG；
c-Myc+clon：F:ttTACTCAAACTGGCTGGGGATG
R:cccagccagtttgagtaAAGATTTGGCTCAATTATATATTTGAAGG；
Klf-4+clon：F:cttTACTCAAACTGGCTGGGGATG
R:aggcgcgccaagcttgcggccgcTTAAAAGTGCCTCTTCATGTGTAAGG。
6. the method of claim 1, wherein the linearized vector is: and (3) carrying out enzyme digestion on the plasmid PB-TRE-hRL, and recovering the PB-TRE skeleton of the vector.
7. Goat-derived OSKM constructed by the method of any one of claims 1 to 6.
8. Use of the method of any one of claims 1 to 6 or the goat-derived OSKM of claim 7 for inducing reprogramming of goat fibroblasts.
9. A method for inducing reprogramming of goat fibroblasts is characterized by comprising the following steps: transferring a reprogramming vector comprising the goat-derived OSKM of claim 7 into goat fibroblasts, and culturing in a medium.
10. The method of claim 9, wherein the reprogramming carrier further comprises: pBase, EF1 alpha, pNhL, hRL and sLargeT plasmids.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116064660A (en) * | 2022-08-22 | 2023-05-05 | 山西农业大学 | Sheep induced pluripotent stem cell and preparation method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104073514A (en) * | 2013-03-27 | 2014-10-01 | 中国农业大学 | Method of preparing pluripotency stem cell |
| CN104762265A (en) * | 2015-04-22 | 2015-07-08 | 扬州大学 | Novel method for obtaining goat-induced multi-potent stem cells |
| CN108676855A (en) * | 2018-07-24 | 2018-10-19 | 内蒙古赛科星家畜种业与繁育生物技术研究院有限公司 | Calibration curve method quantitative fluorescent PCR identifies the method and primer of ox transgene copy number |
| CN114369577A (en) * | 2020-10-15 | 2022-04-19 | 内蒙古大学 | Cattle induced expansion pluripotent adult stem cell, line establishing method and culture solution |
-
2022
- 2022-05-25 CN CN202210576923.6A patent/CN114752625A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104073514A (en) * | 2013-03-27 | 2014-10-01 | 中国农业大学 | Method of preparing pluripotency stem cell |
| CN104762265A (en) * | 2015-04-22 | 2015-07-08 | 扬州大学 | Novel method for obtaining goat-induced multi-potent stem cells |
| CN108676855A (en) * | 2018-07-24 | 2018-10-19 | 内蒙古赛科星家畜种业与繁育生物技术研究院有限公司 | Calibration curve method quantitative fluorescent PCR identifies the method and primer of ox transgene copy number |
| CN114369577A (en) * | 2020-10-15 | 2022-04-19 | 内蒙古大学 | Cattle induced expansion pluripotent adult stem cell, line establishing method and culture solution |
Non-Patent Citations (4)
| Title |
|---|
| JIANGTAO REN 等: "Generation of hircine-induced pluripotent stem cells by somatic cell reprogramming", CELL RESEARCH, vol. 21, no. 5, pages 849 * |
| 刘黎明;孙伟;张金吨;赵丽霞;李树裕;王标;陈杨林;胡树香;吴宝江;李喜和;: "Xist基因抑制对牛SCNT早期胚胎发育的影响", 生物技术通报, no. 01 * |
| 胡广东;郝科兴;黄涛;曾维斌;谷新利;王静;: "绵羊高效转基因通用型piggyBac转座子载体构建及功能验证", 遗传, no. 08 * |
| 赵丽霞;张金吨;张健;李云霞;苏杰;孙伟;赵高平;戴雁峰;郭继彤;胡树香;WANG WEI;LIU PEN-TAO;李喜和;: "利用piggyBac转座子制备牛成体多能干细胞诱导技术研究", 中国生物工程杂志, no. 02 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116064660A (en) * | 2022-08-22 | 2023-05-05 | 山西农业大学 | Sheep induced pluripotent stem cell and preparation method thereof |
| CN116064660B (en) * | 2022-08-22 | 2023-10-17 | 山西农业大学 | Sheep induced pluripotent stem cell and preparation method thereof |
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