CN113278625A - Gene sequence for cultivating specific IGF1 transgenic sheep and identification primer and identification method thereof - Google Patents
Gene sequence for cultivating specific IGF1 transgenic sheep and identification primer and identification method thereof Download PDFInfo
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- 230000009261 transgenic effect Effects 0.000 title claims abstract description 67
- 241001494479 Pecora Species 0.000 title claims abstract description 61
- 101000599951 Homo sapiens Insulin-like growth factor I Proteins 0.000 title claims abstract description 45
- 102100037852 Insulin-like growth factor I Human genes 0.000 title claims abstract description 45
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 24
- 210000004027 cell Anatomy 0.000 claims abstract description 38
- 101150088952 IGF1 gene Proteins 0.000 claims abstract description 23
- 238000001976 enzyme digestion Methods 0.000 claims abstract description 22
- 238000012408 PCR amplification Methods 0.000 claims abstract description 6
- 210000002950 fibroblast Anatomy 0.000 claims abstract description 6
- 238000001962 electrophoresis Methods 0.000 claims description 16
- 108020004999 messenger RNA Proteins 0.000 claims description 15
- 239000002299 complementary DNA Substances 0.000 claims description 13
- 239000012634 fragment Substances 0.000 claims description 10
- 230000014509 gene expression Effects 0.000 claims description 8
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 238000009395 breeding Methods 0.000 claims description 3
- 230000001488 breeding effect Effects 0.000 claims description 3
- 238000010839 reverse transcription Methods 0.000 claims description 3
- 238000010374 somatic cell nuclear transfer Methods 0.000 abstract description 3
- 230000009977 dual effect Effects 0.000 abstract 1
- 210000003491 skin Anatomy 0.000 description 9
- 108020004414 DNA Proteins 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 5
- 239000011492 sheep wool Substances 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 description 4
- 210000001082 somatic cell Anatomy 0.000 description 4
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- 210000001519 tissue Anatomy 0.000 description 3
- 108020004705 Codon Proteins 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
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- 230000015572 biosynthetic process Effects 0.000 description 2
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- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000010449 nuclear transplantation Methods 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
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- 238000002474 experimental method Methods 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
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- 239000013642 negative control Substances 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
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Abstract
The invention provides a gene sequence for cultivating specific IGF1 transgenic sheep, an identification primer and an identification method thereof, wherein the specific IGF1 gene sequence is transfected into sheep fibroblasts, a screened positive transgenic cell line is used as a nuclear donor cell, the specific IGF1 transgenic sheep is obtained by using a conventional somatic cell nuclear transfer mode, and the specific IGF1 transgenic sheep and the situation that the IGF1 gene is expressed by the skin of the specific IGF1 transgenic sheep are identified by using a dual mode of combining a specific primer with PCR amplification and enzyme digestion identification.
Description
Technical Field
The invention relates to the technical field of biological engineering, in particular to a gene sequence for cultivating specific IGF1 transgenic sheep, and an identification primer and an identification method thereof.
Background
Insulin-like growth factor 1 (IGF 1) has a promoting effect on the growth of mammalian stem cells. The growth of sheep wool depends on the growth of hair follicle stem cells, and an exogenous IGF1 gene is transferred into the sheep wool, so that the sheep wool is specifically expressed in skin, the sheep wool can grow quickly, and the sheep wool yield is improved.
The prior art has a phenomenon that after the transgene, a non-homologous exogenous gene is inactivated in a transgenic animal, so that the gene which is the same as the sheep self is transferred to the transgenic animal, the stable expression of the gene is facilitated, and the safety is higher.
The operation is a common operation of IGF1 transgenic sheep, but because the transferred homologous exogenous gene is completely the same as the endogenous gene, the transgenic animal cannot be effectively identified, and whether the IGF1 mRNA expressed by the animal is expressed by the endogenous gene or the exogenous gene cannot be effectively distinguished. Only the relative expression quantity of the gene of transgenic and non-transgenic animal individuals can be compared, and whether the exogenous gene plays a role or not can be deduced, so that the method has contingency and uncertainty.
Disclosure of Invention
Based on the above, it is necessary to provide a gene sequence for breeding specific IGF1 transgenic sheep, and an identification primer and an identification method thereof, aiming at the problem that the prior art cannot distinguish whether the transgenic IGF1 transgenic sheep and the expressed IGF1 mRNA thereof are expressed by endogenous genes or exogenous genes.
A gene sequence for cultivating specific IGF1 transgenic sheep, wherein the gene sequence is shown as SEQ ID NO. 1.
An identification primer for identifying a specific IGF1 transgenic sheep, wherein the specific IGF1 transgenic sheep has a specific IGF1 gene sequence shown as SEQ ID NO.1, and the identification primer sequence is as follows:
F:5'CCAGTCACATCCTCCTCG；
R:5'TTGTTTCCTGCACTCCCT。
a method for identifying a specific IGF1 transgenic sheep having a specific IGF1 gene sequence as shown in SEQ ID No.1 using the identification primer as described above, comprising the steps of:
(1) extracting a cell genome from fibroblasts of a transgenic sheep to be detected;
(2) PCR amplification of the cell genome using the identifying primers as described above;
(3) carrying out enzyme digestion on the amplified cDNA fragment by using Apa I;
(4) and (3) carrying out electrophoresis on the enzyme digestion product, wherein the size of the enzyme digestion product band is 298bp and 76bp, and the enzyme digestion product band is the specific IGF1 transgenic sheep.
In one embodiment, the enzyme digestion reaction system in step (3) is as follows:
a method for identifying a specific IGF1 transgenic sheep skin expressing IGF1 gene using the identification primers described above, said specific IGF1 transgenic sheep having the specific IGF1 gene sequence shown in SEQ ID No.1, comprising the steps of:
(1) adopting skin tissues of transgenic sheep to be detected, extracting total RNA, and carrying out reverse transcription to obtain cDNA;
(2) PCR amplification of the cDNA using the identifying primers as described above;
(3) carrying out enzyme digestion on the amplified cDNA fragment by using Apa I;
(4) carrying out electrophoresis on the enzyme digestion product, and expressing IGF1 mRNA for the transgene sheep endogenous gene when the electrophoresis band is 374 bp; when the electrophoresis bands are 298bp and 76bp, IGF1 mRNA is expressed as an exogenous gene; when 3 bands are present in the electrophoretic band, IGF1 mRNA is expressed simultaneously as an endogenous gene and an exogenous gene.
In one embodiment, the enzyme digestion reaction system in step (3) is as follows:
compared with the prior art, the invention has the following advantages:
the invention relates to a gene sequence for cultivating specific IGF1 transgenic sheep, an identification primer and an identification method thereof, wherein a synthesized specific IGF1 gene sequence is transfected to sheep fibroblasts, a screened positive transgenic cell line is used as a nuclear donor cell, a conventional somatic cell nuclear transplantation mode is used for obtaining the specific IGF1 transgenic sheep, and the specific IGF1 transgenic sheep and the situation of the skin expression IGF1 gene thereof are quickly, intuitively and accurately identified by combining a double mode of PCR amplification and enzyme digestion identification with a specific primer.
Drawings
FIG. 1 is a schematic diagram of a sheep skin-specific expression IGF1 gene DNA fragment;
FIG. 2 is an enzyme-cleaved map of the genome of the transgenic somatic cell of example 1 (wherein M: 150bp DNA Ladder, 1: not cleaved PCR product, 2: negative control, 3: false positive, 4: heterozygote, 5: heterozygote, 6: positive control).
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, the present invention is described in detail with reference to the following embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
To achieve the object of the present invention, the present invention designs a DNA sequence, specifically, a DNA sequence which is substantially identical to the published sheep IGF1 gene (NM-001009774.3) at positions 99-563 in the 5 'to 3' direction, but is changed from adenine (A) to cytosine (C) at amino acid 150 from the ATG initiation codon. According to the degeneracy of codons, the amino acids expressed by the specific IGF1 gene sequence designed by the invention are not changed and are glycine (codons: GGA and GGC), but an Apa I enzyme cutting site (GGGCCC) is manufactured in the specific IGF1 gene sequence designed by the invention. Therefore, the product obtained by PCR technique using sheep genome as template and specific primer (F:5 'CCAGTCACATCCTCCTCG; R:5' TTGTTTCCTGCACTCCCT) can be identified by Apa I digestion. The Apa I enzyme can cut transgenic sheep, and conversely non-transgenic sheep. Similarly, the product obtained by using cDNA reverse transcribed from transgenic sheep mRNA as a template and using a PCR technology through a specific primer (F:5 'CCAGTCACATCCTCCTCG; R:5' TTGTTTCCTGCACTCCCT) can be identified by Apa I enzyme, wherein the Apa I enzyme can cut the product to express exogenous genes, and conversely, the product can express endogenous genes.
1. Design of specific sheep IGF1 Gene sequences
Sheep IGF1 gene (NM-001009774.3) published sequence as follows:
ATGGGAAAAATCAGCAGTCTTCCAACCCAATTATTTAAGTGCTGCTT TTGTGATTTCTTGAAGCAGGTGAAGATGCCAGTCACATCCTCCTCGCATCT CTTCTATCTGGCCCTGTGCTTGCTCGCCTTCAGCAGCTCTGCCACGGCGGGACCTGAGACCCTCTGCGGGGCTGAGTTGGTGGATGCTCTCCAGTTCGTGT GCGGAGACAGGGGCTTTTATTTCAACAAGCCCACGGGGTACGGCTCGAGC AGTCGGAGGGCGCCCCAGACAGGAATCGTGGATGAGTGCTGCTTCCGGA GCTGTGATCTGAGGAGGCTGGAGATGTACTGTGCGCCTCTCAAGGCCGCC AAGTCGGCCCGCTCAGTCCGTGCCCAGCGCCACACCGACATGCCCAAGGC TCAGAAGGAAGTACATTTGAAGAACACAAGCAGAGGGAGTGCA GGAAACAAGAACTACAGAATGTAG
the specific IGF1 gene sequence SEQ ID NO.1 designed by the invention is as follows:
ATGGGAAAAATCAGCAGTCTTCCAACCCAATTATTTAAGTGCTGCTT TTGTGATTTCTTGAAGCAGGTGAAGATGCCAGTCACATCCTCCTCGCATCT CTTCTATCTGGCCCTGTGCTTGCTCGCCTTCAGCAGCTCTGCCACGGCGGGCCCTGAGACCCTCTGCGGGGCTGAGTTGGTGGATGCTCTCCAGTTCGTGT GCGGAGACAGGGGCTTTTATTTCAACAAGCCCACGGGGTACGGCTCGAGC AGTCGGAGGGCGCCCCAGACAGGAATCGTGGATGAGTGCTGCTTCCGGA GCTGTGATCTGAGGAGGCTGGAGATGTACTGTGCGCCTCTCAAGGCCGCC AAGTCGGCCCGCTCAGTCCGTGCCCAGCGCCACACCGACATGCCCAAGGC TCAGAAGGAAGTACATTTGAAGAACACAAGCAGAGGGAGTGCA GGAAACAAGAACTACAGAATGTAG
2. obtaining kap 6.1.1 promoter sequence
The sheep Kap6.1 promoter sequence (M95719) was obtained from a review.
3. Obtaining of sheep skin specific expression IGF1 gene DNA fragment
The corresponding DNA sequences were synthesized in the order shown in FIG. 1.
4. Obtaining and identifying transgenic somatic cells
The synthetic DNA sequence was transfected into sheep fibroblasts by electrotransfection. Positive cells were selected as follows:
the cell genome was extracted, and the desired fragment (374bp) was amplified using the following sequence as an identifying primer (annealing temperature 48 ℃).
F:5'CCAGTCACATCCTCCTCG；
R:5'TTGTTTCCTGCACTCCCT
The amplified fragment was digested with Apa I:
and then carrying out electrophoresis on the enzyme digestion product, wherein cell lines with the size of the enzyme digestion product bands of 298bp and 76bp are positive transgenic cell lines, only one band is a false positive cell line, and the cell line without the band is a negative cell line.
5. Acquisition of transgenic sheep
The positive transgenic cell line is used as a nuclear donor cell, and the transgenic sheep is obtained by using a conventional somatic cell nuclear transfer mode.
6. Identification of transgenic sheep and identification of skin-expressed IGF1 gene
The identification method of the transgenic sheep is the same as the identification method of the transgenic cells.
And (3) extracting total RNA from positive transgenic sheep skin tissues, carrying out reverse transcription on the total RNA to obtain cDNA, and identifying by using the cDNA as a template according to the method for identifying positive cells. When the electrophoresis band is 374bp, IGF1 mRNA is expressed by the endogenous gene of the transgenic sheep; when the electrophoresis bands are 298bp and 76bp, IGF1 mRNA is expressed as an exogenous gene; when 3 bands exist in the electrophoresis band, IGF1 mRNA is expressed simultaneously as an endogenous gene and an exogenous gene.
The invention is further illustrated by the following examples.
Example 1
(1) Synthetic sheep skin specific expression IGF1 gene expression sequence
Taking FIG. 1 as an example, the expression sequence SEQ ID NO.2 was synthesized as follows:
CTGCAGTTCATGGGGTCACTAAGAGTCGGGCATGGCTGAGCGACTTCACTTTCATGTATCACTTTCAT GCATTGGAGAAGGAAATGGCAACGCACTCCAGTGTTCTTGCCTGGAGAATCCCAGGGCTGGGGGAGCCTGGTGCAC TGCCATCTCTGGGGTCGCACAGAGTCGGACATGACTGAAGAGACTTAGCAGCAGCAGTAGCAGCATGTTGATAAGG GACTTGGTTTAGCACATTAATAAACATAAATATGTTAGTATATTGGATATTTTCTTAGAATATAAATCTAACACTA ATGAACAGACTAGTTTGTATAACTGTATATTCAATTTAGAAAAACAAGTGGAGAAATCAGATTTCAAGAAATAACT CCTTTTTGCAGTCCTTCAATAGAAATTGAGCATAAATGTGAATTAGTCATTGGCATAGACAGAAAAATATAATGCA TTTTGCTCAGACTTGGTTTACTGGAAACTTTAACTGGTTGGATTATGATCAACATCATGGGAATAAAAGATACATT GTAGTTTCAATATAGGAAAGAAACTGAATCACTGAAGAAGATAATTTGGATCAAGAAGATAAGAATCTTTGAGTAA AAAGGAGTTGTTAGTCTTAAGAAAAAAATTTTAACGTTTGGTGAAACAAACTGAGGTCAAGAGCAAATAAGATTAA GACCAACAAATATATTTCTCACTATACTGAAGGTGCTAGGTGGTTAAAATAAAATGTGTGATCTGGGACAGGACTG TGTAGGTGTGAGTCTGCATCTCCTCTCATTCAATTCCTTAACTGGATAAGAGGAATCTAAACTGAGATGTCAACAC AGCAAGCCTGCTGAATTTCTCTGAGGTTTCATCTTTGGTTGTGAACAACAAGCTAATTAGTCCAGTCATAAAGTTA GCCAATGGCATGAAGGTGTGGTGGGTCACACCCACACTGAGAGCATATAAAAGGCCCTCTGCAGGGAGAAATGTCC ACACTCAAGTGACACTTCTACTCTAATTCTCTACCCGAGAACAACCTCAACAAGCAACACCTCTAGAATGGGAAAAATCAGCAGTCT TCCAACCCAATTATTTAAGTGCTGCTTTTGTGATTTCTTGAAGCAGGTGAA GATGCCAGTCAC ATCCTCCTCGCATCTCTTCTATCTGGCCCTGTGCTTGCT CGCCTTCAGCAGCTCTGCCACGGCGGGACCTGAGACCCTCTGCGGGGCTG AGTTGGTGGATGCTCTCCAGTTCGTGTGCGGAGACAGGGGCTTTTATTTC AACAAGCCCACGGGGTACGGCTCGAGCAGTCGGAGGGCGCCCCAGACAG GAATCGTGGATGAGTGCTGCTTCCGGAGCTGTGATCTGAGGAGGCTGGAG ATGTACTGTGCGCCTCTCAAGGCCGCCAAGTCGGCCCGCTCAGTCCGTGC CCAGCGCCACACCGACATGCCCAAGGCTCAGAAGGAAGTACATTTGAAG AACACAAGCAGAGGGAGTGCAGGAAACAAGAACTACAGAATGTAGTTTT TT
(2) obtaining and identifying transgenic somatic cells
The synthesized DNA fragment is transferred into a sheep fibroblast by a Lonza electrotransfer instrument, and the specific procedure is as follows: mu.g of Kap6.1-IGF1 plasmid was added to 100. mu.L of the electrotransfer, pipetted well and mixed 2X 106Suspending individual cell, transferring into electric rotary cup, performing electric rotation by CZ-167 procedure, standing for 10min, inoculating cell into 10cm culture dish, and culturing at 37 deg.C and 5% CO2And culturing overnight under the saturated humidity condition, selecting single cells after 12 hours, putting the single cells into a 96-well plate for single cell culture, taking partial cells for identification after the cells form clones, and using the positive cells for nuclear transplantation experiments.
Positive cells were selected as follows:
extracting cell genome with the kit, identifying the primer with the sequence, and amplifying the target segment
(374bp)。
F: 5' CCAGTCACATCCTCCTCG；
R: 5' TTGTTTCCTGCACTCCCT；
Reaction system: the lysate was 15. mu.L, 10. mu. mol/L F, 2. mu.L each of R primers, 2 XPCR mix 25. mu.L, ddH2O supplemented to 50. mu.L. The reaction conditions are as follows: 95 ℃ for 5 min, 35 cycles (95 ℃ for 30s, 48 ℃ for 30s, 72 ℃ for 30 s), 72 ℃ for 10 min.
The amplified fragment was digested with Apa I:
and then carrying out electrophoresis on the enzyme digestion product, wherein cell lines with the band sizes of 298bp and 76bp of the enzyme digestion product are positive transgenic cell lines, only one cell line is a false positive cell line, the cell line without the band is a negative cell line, and the electrophoresis condition is shown in figure 2.
(3) Acquisition of transgenic sheep
The positive transgenic cell line is used as a nuclear donor cell, and the transgenic sheep is obtained by using a conventional somatic cell nuclear transfer mode.
(4) Identification of transgenic sheep and identification of skin-expressed IGF1 gene
The identification method of the transgenic sheep is the same as the identification method of the transgenic cells. The positive transgenic sheep skin tissue is adopted to extract total RNA, the total RNA is reversely transcribed into cDNA, the cDNA is taken as a template, and the identification is carried out by a positive cell identification method. When the electrophoresis band is 374bp, IGF1 mRNA is expressed by the endogenous gene of the transgenic sheep; when the electrophoresis bands are 298bp and 76bp, IGF1 mRNA is expressed as an exogenous gene;
when 3 bands are present in the electrophoretic band, IGF1 mRNA is expressed simultaneously as an endogenous gene and as an exogenous gene.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> academy of agricultural reclamation of Sinkiang
<120> gene sequence for breeding specific IGF1 transgenic sheep, and identification primer and identification method thereof
<130> 2021.3.1
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aagcaggtga agatgccagt cacatcctcc tcgcatctct tctatctggc cctgtgcttg 120
ctcgccttca gcagctctgc cacggcgggc cctgagaccc tctgcggggc tgagttggtg 180
gatgctctcc agttcgtgtg cggagacagg ggcttttatt tcaacaagcc cacggggtac 240
ggctcgagca gtcggagggc gccccagaca ggaatcgtgg atgagtgctg cttccggagc 300
tgtgatctga ggaggctgga gatgtactgt gcgcctctca aggccgccaa gtcggcccgc 360
tcagtccgtg cccagcgcca caccgacatg cccaaggctc agaaggaagt acatttgaag 420
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ctgcagttca tggggtcact aagagtcggg catggctgag cgacttcact ttcatgtatc 60
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cagggctggg ggagcctggt gcactgccat ctctggggtc gcacagagtc ggacatgact 180
gaagagactt agcagcagca gtagcagcat gttgataagg gacttggttt agcacattaa 240
taaacataaa tatgttagta tattggatat tttcttagaa tataaatcta acactaatga 300
acagactagt ttgtataact gtatattcaa tttagaaaaa caagtggaga aatcagattt 360
caagaaataa ctcctttttg cagtccttca atagaaattg agcataaatg tgaattagtc 420
attggcatag acagaaaaat ataatgcatt ttgctcagac ttggtttact ggaaacttta 480
actggttgga ttatgatcaa catcatggga ataaaagata cattgtagtt tcaatatagg 540
aaagaaactg aatcactgaa gaagataatt tggatcaaga agataagaat ctttgagtaa 600
aaaggagttg ttagtcttaa gaaaaaaatt ttaacgtttg gtgaaacaaa ctgaggtcaa 660
gagcaaataa gattaagacc aacaaatata tttctcacta tactgaaggt gctaggtggt 720
taaaataaaa tgtgtgatct gggacaggac tgtgtaggtg tgagtctgca tctcctctca 780
ttcaattcct taactggata agaggaatct aaactgagat gtcaacacag caagcctgct 840
gaatttctct gaggtttcat ctttggttgt gaacaacaag ctaattagtc cagtcataaa 900
gttagccaat ggcatgaagg tgtggtgggt cacacccaca ctgagagcat ataaaaggcc 960
ctctgcaggg agaaatgtcc acactcaagt gacacttcta ctctaattct ctacccgaga 1020
acaacctcaa caagcaacac ctctagaatg ggaaaaatca gcagtcttcc aacccaatta 1080
tttaagtgct gcttttgtga tttcttgaag caggtgaaga tgccagtcac atcctcctcg 1140
catctcttct atctggccct gtgcttgctc gccttcagca gctctgccac ggcgggacct 1200
gagaccctct gcggggctga gttggtggat gctctccagt tcgtgtgcgg agacaggggc 1260
ttttatttca acaagcccac ggggtacggc tcgagcagtc ggagggcgcc ccagacagga 1320
atcgtggatg agtgctgctt ccggagctgt gatctgagga ggctggagat gtactgtgcg 1380
cctctcaagg ccgccaagtc ggcccgctca gtccgtgccc agcgccacac cgacatgccc 1440
aaggctcaga aggaagtaca tttgaagaac acaagcagag ggagtgcagg aaacaagaac 1500
tacagaatgt agtttttt 1518
Claims (6)
1. A gene sequence for breeding specific IGF1 transgenic sheep, which is characterized in that the gene sequence is shown as SEQ ID NO. 1.
2. An identification primer for identifying a specific IGF1 transgenic sheep, wherein the specific IGF1 transgenic sheep has a specific IGF1 gene sequence shown as SEQ ID NO.1, and the identification primer sequence is as follows:
F:5'CCAGTCACATCCTCCTCG；
R:5'TTGTTTCCTGCACTCCCT。
3. a method for identifying a specific IGF1 transgenic sheep having a specific IGF1 gene sequence as shown in SEQ ID No.1 using the identification primer set forth in claim 2, comprising the steps of:
(1) extracting a cell genome from fibroblasts of a transgenic sheep to be detected;
(2) performing PCR amplification of the genome of the cell using the identifying primers of claim 2;
(3) carrying out enzyme digestion on the amplified cDNA fragment by using Apa I;
(4) and (3) carrying out electrophoresis on the enzyme digestion product, wherein the size of the enzyme digestion product band is 298bp and 76bp, and the enzyme digestion product band is the specific IGF1 transgenic sheep.
4. The method for identifying a specific IGF1 transgenic sheep using the identification primer as claimed in claim 2, wherein the enzyme digestion reaction system in step (3) is as follows:
5. a method for identifying a specific IGF1 transgenic sheep skin expressing IGF1 gene using the identification primer set forth in claim 2, said specific IGF1 transgenic sheep having the specific IGF1 gene sequence shown in SEQ ID No.1, the method comprising the steps of:
(1) adopting skin tissues of transgenic sheep to be detected, extracting total RNA, and carrying out reverse transcription to obtain cDNA;
(2) performing PCR amplification on the cDNA using the identifying primer of claim 2;
(3) carrying out enzyme digestion on the amplified cDNA fragment by using ApaI;
(4) carrying out electrophoresis on the enzyme digestion product, and expressing IGF1 mRNA for the transgenic sheep endogenous gene when the electrophoresis band is 374 bp; when the electrophoresis bands are 298bp and 76bp, IGF1 mRNA is expressed as an exogenous gene; when 3 bands are present in the electrophoretic band, IGF1 mRNA is expressed simultaneously as an endogenous gene and a foreign gene.
6. The method for identifying specific IGF1 transgenic sheep skin expression IGF1 gene according to claim 5 by using the identification primer as described in claim 2, wherein the enzyme digestion reaction system in step (3) is as follows:
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Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1873001A (en) * | 2005-04-21 | 2006-12-06 | 李宁 | Method for procducing great cattles through transgene cloning ferritin gene of human milk |
| CN101503706A (en) * | 2009-03-09 | 2009-08-12 | 刘红林 | Method for obtaining transgenic chicken by lentivirus vector mediated in vivo transfection of spermatogonial stem cell |
| CN102041271A (en) * | 2010-11-16 | 2011-05-04 | 南京农业大学 | Mammary gland specificity expression vector capable of efficiently expressing para-insulin growth factor IGF-1 |
| CN102978202A (en) * | 2012-10-10 | 2013-03-20 | 中国农业科学院北京畜牧兽医研究所 | Over-expression vector for muscle specific expression of pig IGF1 gene |
| CN103255168A (en) * | 2013-05-06 | 2013-08-21 | 深圳华大基因研究院 | Construct and application thereof |
| CN103266174A (en) * | 2013-05-22 | 2013-08-28 | 青岛农业大学 | PCR (Polymerase Chain Reaction) primer and identification method of athetis lepigone and sibling species thereof |
| CN108611354A (en) * | 2018-05-11 | 2018-10-02 | 中国农业大学 | A kind of method for identifying molecules that FT mRNA molecules transmit between congener anvil fringe |
| CN110894542A (en) * | 2019-12-31 | 2020-03-20 | 扬州大学 | Primer for identifying types of GS5 gene and GLW7 gene of rice and application of primer |
| CN111560372A (en) * | 2020-05-07 | 2020-08-21 | 海南波莲水稻基因科技有限公司 | Expression cassette and vector for restoring male fertility of rice OsCYP704B2 mutant, and detection method and application thereof |
| CN111593135A (en) * | 2020-05-07 | 2020-08-28 | 海南波莲水稻基因科技有限公司 | Detection primer and method for identifying internal and external genes in transgenic material and selfing, hybridization and backcross progeny thereof |
-
2021
- 2021-03-12 CN CN202110270295.4A patent/CN113278625A/en active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1873001A (en) * | 2005-04-21 | 2006-12-06 | 李宁 | Method for procducing great cattles through transgene cloning ferritin gene of human milk |
| CN101503706A (en) * | 2009-03-09 | 2009-08-12 | 刘红林 | Method for obtaining transgenic chicken by lentivirus vector mediated in vivo transfection of spermatogonial stem cell |
| CN102041271A (en) * | 2010-11-16 | 2011-05-04 | 南京农业大学 | Mammary gland specificity expression vector capable of efficiently expressing para-insulin growth factor IGF-1 |
| CN102978202A (en) * | 2012-10-10 | 2013-03-20 | 中国农业科学院北京畜牧兽医研究所 | Over-expression vector for muscle specific expression of pig IGF1 gene |
| CN103255168A (en) * | 2013-05-06 | 2013-08-21 | 深圳华大基因研究院 | Construct and application thereof |
| CN103266174A (en) * | 2013-05-22 | 2013-08-28 | 青岛农业大学 | PCR (Polymerase Chain Reaction) primer and identification method of athetis lepigone and sibling species thereof |
| CN108611354A (en) * | 2018-05-11 | 2018-10-02 | 中国农业大学 | A kind of method for identifying molecules that FT mRNA molecules transmit between congener anvil fringe |
| CN110894542A (en) * | 2019-12-31 | 2020-03-20 | 扬州大学 | Primer for identifying types of GS5 gene and GLW7 gene of rice and application of primer |
| CN111560372A (en) * | 2020-05-07 | 2020-08-21 | 海南波莲水稻基因科技有限公司 | Expression cassette and vector for restoring male fertility of rice OsCYP704B2 mutant, and detection method and application thereof |
| CN111593135A (en) * | 2020-05-07 | 2020-08-28 | 海南波莲水稻基因科技有限公司 | Detection primer and method for identifying internal and external genes in transgenic material and selfing, hybridization and backcross progeny thereof |
Non-Patent Citations (6)
| Title |
|---|
| CHUNLI WANG等: "Porcine IGF-1R synonymous mutations in the intracellular domain affect cell proliferation and alter kinase activity", 《INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES》 * |
| LINDA MONDINI等: "Assessing Plant Genetic Diversity by Molecular Tools", 《DIVERSITY》 * |
| MEIRA AN等: "Ovis aries insulin like growth factor 1 (IGF1), mRNA", 《GENBANK DATABASE》 * |
| 生吉萍等主编: "《普通高等教育"十三五"规划教材 高等学校专业教材 食品基因工程导论》", 28 February 2017, 中国轻工业出版社 * |
| 赵 帅等: "实时定量 PCＲ 检测转基因绵羊拷贝数", 《新疆农业科学》 * |
| 郭旭东等: "绒山羊表达红色荧光及转胰岛素样生长因子Ⅰ基因体细胞核移植胚胎的制备", 《中国科学(C辑:生命科学)》 * |
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