CN102943092B - General type PiggyBac transposon transgenosis carrier and preparation method thereof - Google Patents
General type PiggyBac transposon transgenosis carrier and preparation method thereof Download PDFInfo
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- CN102943092B CN102943092B CN201210472708.8A CN201210472708A CN102943092B CN 102943092 B CN102943092 B CN 102943092B CN 201210472708 A CN201210472708 A CN 201210472708A CN 102943092 B CN102943092 B CN 102943092B
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Abstract
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Claims (1)
- One kind universal piggyBactransposon transgene carrier, is characterized in that, comprises donor carrier and assistant carrier;Described donor carrier comprises piggyBactransposon 5 ' repeats end sequence 5 ' TR and 3 ' and repeats end sequence 3 ' TR, is provided with two insulators between 5 ' TR and 3 ' TR, between insulator sequence, is provided with multiple clone site; The nucleotide sequence of 5 ' described TR is as shown in SEQ.ID.NO.1; The nucleotide sequence of 3 ' TR is as shown in SEQ.ID.NO.2;Between the insulator of close 5 ' TR and multiple clone site, be also provided with interconnective antibiotic-screening gene and fluorescent mark gene; Described antibiotic-screening gene and the two ends of fluorescent mark gene are respectively equipped with two Loxp sequences in the same way: Loxp1 and Loxp2;Between Loxp1 and antibiotic-screening gene, be also provided with SV40 PloyA, antibiotic-screening gene is connected by 2A sequence with fluorescent mark gene, is also provided with CMV promotor between fluorescent mark gene and Loxp2; Described 2A sequence is: cctaacgccg gagaagaaga agggcccagg gttggactca acgtctcctg ccaacttgag aaggtcaaaa tt;The nucleotide sequence of described insulator is as shown in SEQ.ID.NO.4, and the nucleotide sequence of described multiple clone site is as shown in SEQ.ID.NO.3;Described assistant carrier comprises piggyBactransposase encoding gene Pbase, is provided with promotor in the upstream of Pbase, is provided with PloyA in its downstream.2 .as claimed in claim 1 universal piggyBactransposon transgene carrier, is characterized in that, the nucleotide sequence of described Pbase is as shown in SEQ.ID.NO.5; Described promotor is PTK promotor.3 .as claimed in claim 1 universal piggyBactransposon transgene carrier, is characterized in that, described antibiotic-screening gene is Neo gene, and described fluorescent mark gene is EGFP.4 .a kind of universal piggyBacthe preparation method of transposon transgene carrier, is characterized in that, comprises the following steps:1) synthesize respectively as shown in SEQ.ID.NO.1 piggyBactransposon 5 ' TR and as shown in SEQ.ID.NO.2 piggyBactransposon 3 ' TR sequence, and chicken source insulator sequence as shown in SEQ.ID.NO.4, as shown in SEQ.ID.NO.3 the sequence of multiple clone site, then according to 5 ' insulator-multiple clone site sequence-chicken source, TR-chicken source insulator- piggyBacthe element of transposon 3 ' TR is linked in sequence, and the mode that constructed fragment is cloned by TA is cloned in pMD18-T simple carrier, obtains plasmid TP;2) the NEO gene that amplification upstream is connected with 2A sequence is cloned into the downstream of EGFP in pEGFP-C1 plasmid, obtains plasmid pNA; Described 2A sequence is: cctaacgccg gagaagaaga agggcccagg gttggactca acgtctcctg ccaacttgag aaggtcaaaa tt;3) taking pNA as template, the fragment between primer amplification CMV to PloyA taking CEANF and CEANR, and increased fragment is cloned between the insulator and multiple clone site near 5 ' end in TP plasmid, obtain pBNW-TP1 carrier;Described CEANF is: gagcagatct ataacttcgt atagcataca ttatacgaag ttattgcgtt atcccctgat tctgt;Described CEANR is: tatcgtcgac ataacttcgt ataatgtatg ctatacgaag ttatcgctta caatttacgc cttaag;4) amplification PTK promotor, and be cloned in pEGFP-C1 plasmid, pPTK plasmid obtained;5) the Pbase encoding gene of amplification as shown in SEQ.ID.NO.5, and be cloned into the downstream of PTK promotor in pPTK plasmid, obtain plasmid pPTK-Pbase;6) taking pPTK-PBase as template, the fragment of amplification between PTK to PloyA, and the mode that increased fragment is cloned by TA connects and is cloned in pMD18-T simple carrier, obtains pBNW-TD2 carrier;The common composition of constructed carrier pBNW-TP1 and pBNW-TD2 is universal piggyBactransposon transgene carrier.5 .claimed in claim 1 universal piggyBacthe application of transposon transgene carrier in external source gene transfecting cell: by exogenous gene cloning to the multiple clone site of donor carrier, and then with assistant carrier mixing transfectional cell, stable screening cell monoclonal.
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Families Citing this family (10)
Publication number | Priority date | Publication date | Assignee | Title |
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CN103509812B (en) * | 2013-08-07 | 2016-01-27 | 华南农业大学 | The specific transgene carrier of salivary organization and construction process thereof |
CN103695451B (en) * | 2013-08-07 | 2016-04-06 | 华南农业大学 | The transgene carrier of salivary gland tissue specific expression foreign protein and transgenic pig and construction process thereof |
CN103923941B (en) * | 2014-04-29 | 2016-08-24 | 西南大学 | Realize piggyBac carrier of silkworm transgenic long-time stability and preparation method thereof |
CN105154473B (en) * | 2015-09-30 | 2019-03-01 | 上海细胞治疗研究院 | A kind of transposon integration system of highly effective and safe and application thereof |
MX2018007203A (en) * | 2015-12-14 | 2018-11-12 | Genomefrontier Therapeutics Inc | Transposon system, kit comprising the same, and uses thereof. |
CN108753727A (en) * | 2018-06-11 | 2018-11-06 | 上海科技大学 | A kind of GPCR targeted drugs screening system and its structure and application |
CN109628488A (en) * | 2018-12-28 | 2019-04-16 | 赛业(苏州)生物科技有限公司 | The method for constructing gene overexpression based on piggyBAC system or interfering stable cell strain |
CN110551693A (en) * | 2019-07-22 | 2019-12-10 | 佛山普津生物技术有限公司 | Method for screening HEK cells by antibiotics |
CN114317600A (en) * | 2020-10-12 | 2022-04-12 | 上海君赛生物科技有限公司 | Novel PiggyBac transposon system and application thereof |
TW202428609A (en) * | 2023-01-05 | 2024-07-16 | 大陸商上海齊魯製藥研究中心有限公司 | Truncated insulator and application thereof |
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CN101297031A (en) * | 2005-05-14 | 2008-10-29 | 复旦大学 | PiggyBac used as genetic operation and analysis tool of vertebrate |
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US8399643B2 (en) * | 2009-02-26 | 2013-03-19 | Transposagen Biopharmaceuticals, Inc. | Nucleic acids encoding hyperactive PiggyBac transposases |
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Patent Citations (3)
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CN1860235A (en) * | 2003-07-28 | 2006-11-08 | 奥西泰克有限公司 | Expression systems for insect pest control |
CN101297031A (en) * | 2005-05-14 | 2008-10-29 | 复旦大学 | PiggyBac used as genetic operation and analysis tool of vertebrate |
CN101343638A (en) * | 2008-08-20 | 2009-01-14 | 中国人民解放军军事医学科学院军事兽医研究所 | Toxoplasma transgenosis carrier based on piggyback transposon and construction method |
Non-Patent Citations (2)
Title |
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PiggyBac Transposon-Mediated, Reversible Gene Transfer in Human Embryonic Stem Cells;You-Tzung Chen等;《STEM CELLS AND DEVELOPMENT》;20100630;第19卷(第6期);第766页左栏第二段,第766页右栏第1-3段,参考文献36 * |
You-TzungChen等.PiggyBacTransposon-Mediated Reversible Gene Transfer in Human Embryonic Stem Cells.《STEM CELLS AND DEVELOPMENT》.2010 * |
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Inventor after: Zhang Yong Inventor after: Hu Guangdong Inventor after: Wang Jing Inventor after: Yu Yuan Inventor after: Gao Yuanpeng Inventor before: Hu Guangdong Inventor before: Zhang Yong Inventor before: Wang Jing Inventor before: Yu Yuan Inventor before: Gao Yuanpeng |
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Free format text: CORRECT: INVENTOR; FROM: HU GUANGDONG ZHANG YONG WANG JING YU YUAN GAO YUANPENG TO: ZHANG YONG HU GUANGDONG WANG JING YU YUAN GAO YUANPENG |
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