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Composite piggybac recombinant vector and its preparation method and application

A recombinant vector and composite technology, which can be used in recombinant DNA technology, introduction of foreign genetic material using vectors, animal husbandry, etc., and can solve problems such as lepidopteran species that have not yet been applied

Inactive Publication Date: 2017-10-20
SOUTHWEST UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this strategy to achieve stable transgene integration by deleting piggyBac flanking sequences has not been applied to silkworms and other Lepidoptera species so far.

Method used

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  • Composite piggybac recombinant vector and its preparation method and application
  • Composite piggybac recombinant vector and its preparation method and application
  • Composite piggybac recombinant vector and its preparation method and application

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Embodiment Construction

[0054] The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. For the experimental methods not specified in the examples, the conventional conditions are generally followed, such as the conditions described in the Molecular Cloning Experiment Guide (Third Edition, J. Sambrook et al.), or the conditions suggested by the manufacturer.

[0055] The practical diapause silkworm strain 871 (white cocoon) of the Chinese lineage is preserved in the laboratory of the inventor. Vector pSLfa1180fa(Carsten Horn.et al2000), pBac{3×P3-DsRedaf}(AiChun Zhao.et al2010), pSL{3×P3-EGFP-SV40}(Dingpei Long.et al2012), pHA3PIG(Toshiki Tamura.et al2000), pMLS104 (Mark L. Siegal. et al1996) and pBac{3×P3-DsRedaf}-R3 (AiChun Zhao. et al2010) were all preserved by the inventor's laboratory.

[0056] 1. Construction of composite piggyBac recombinant vector

[0057] Composite piggyBac recombinant vector pBac{R1-3×P3-DsRed-SV40-L...

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Abstract

The invention discloses a composite piggyBac recombinant vector and its preparation method and application. The recombinant vector is inserted between the wild-type piggyBac transposon arms R1 and L1 with a target gene expression cassette, screening marker gene expression cassettes I and II, The piggyBac transposase expression cassette regulated by the heat shock promoter and the truncated piggyBac transposon arms L2 and R2 can conveniently mediate the integration of the target gene together with the screening marker gene and the piggyBac transposase expression cassette sequence in the silkworm genome , establish and screen transgenic silkworm lines with high expression of the target gene, and then induce the expression of piggyBac transposase in the transgenic silkworm by heat shock treatment, which can realize the complete deletion of all transposon sequences and screening marker gene sequences in the offspring transgenic silkworm genome, The rest of the target gene expression cassette anchored by the attP site can easily realize the replacement of other foreign genes with the target gene through the cassette exchange reaction mediated by phiC31 integrase, and then realize the replacement of other foreign genes at this genomic site. fixed-point integration.

Description

technical field [0001] The invention belongs to the field of biotechnology, relates to a piggyBac recombination vector and a preparation method thereof, and also relates to the application of the recombination vector in preparing a stable, replaceable and high-efficiency transgenic silkworm expression system. Background technique [0002] Since the first report of insect genetic transformation mediated by P factor in Drosophila melanogaster in 1982, the transposon-based genetic transformation system has been widely used in insects and other invertebrates over the past 30 years. and applied research. Among them, the most widely used and mature transposon system is the piggyBac transposon derived from Trichoplusia ni. piggyBac is a typical type II transposon system, and its internal region sequence encodes a transposase with 594 amino acids. The transposase can act on the terminal inverted repeat (Terminal inverted repeat, TIR) at both ends of the piggyBac transposon and its...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/66C12N15/85A01K67/04
Inventor 赵爱春龙定沛陆威健鲁成向仲怀
Owner SOUTHWEST UNIV
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