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Efficient transgenosis method with mediation of transcription activator-like effector protein

A transcriptional activation and effector protein technology, applied in the field of transgenics, can solve the problems of low transgenic efficiency, low transgenic efficiency, and increased workload of researchers, saving manpower and material resources and simplifying the process

Active Publication Date: 2015-05-27
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the transgenic efficiency mediated by piggyBac transposons is still low, and the transformation efficiency is unstable. In recent years, the average transgenic efficiency in silkworms is only 8.8%.
Therefore, the low transgenic efficiency has added a great workload to researchers, which largely limits the further promotion and application of this technology.

Method used

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  • Efficient transgenosis method with mediation of transcription activator-like effector protein
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  • Efficient transgenosis method with mediation of transcription activator-like effector protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] For the purpose of improving the transgenic efficiency of animals, a new type of helper plasmid ( figure 1 ). The specific method is: first assemble the TALE sequence (such as SEQ ID NO.3); connect a PBase sequence to the 3' end (COON end) of the TALE, which is in the same coding frame as the previous TALE sequence, and is used to express TALE-PBase Fusion protein, there is a 209bp linker sequence (Linker) (such as SEQ ID NO.4) between TALE and PBase; at the 5' end of TALE (NH 2 end) to construct a SP6 prokaryotic promoter (such as SEQ ID NO.1), which can transcribe downstream genes into mRNA in vitro; a nuclear localization sequence (NLS) (such as SEQ ID NO.2) was also constructed, using To guide TALE-PBase fusion protein into the nucleus.

[0022] At the same time, a piggyBac transposon plasmid DNA (such as SEQ ID NO.5) containing an expression cassette ending in 3×P3-EGFP-SV40 Poly A was also constructed ( figure 2 ), wherein ITR is the inverted repeat sequence o...

Embodiment 2

[0027] The construction method of the TALE-PBase fusion protein expression vector and the transposon plasmid DNA comprising the 3×P3-EGFP expression cassette is as in Example 1, the helper plasmid of Example 2 and the transposon plasmid comprising the 3×P3-EGFP expression cassette The DNA is the same as in Example 1.

[0028] The novel helper plasmid of the present invention was transcribed into mRNA in vitro, and then dissolved together with 3×P3-EGFP transposon plasmid DNA in pH 7.6 phosphate buffer without RNase. The concentration of 3×P3-EGFP transposon plasmid DNA in phosphate buffer was 300ng / μL, and the concentration of novel helper plasmid mRNA was 200ng / μL.

[0029] Using silkworm as a model organism, the mixed DNA and mRNA solution was introduced into the fertilized eggs of P50 silkworm within 4 hours after laying eggs by microinjection, and the total injection volume was about 10 nl. The injected silkworm eggs were reared to adults (G0 generation) under the conditi...

Embodiment 3

[0032] The construction method of the TALE-PBase fusion protein expression vector is as in Example 1. At the same time, a piggyBac transposon plasmid containing two expression cassettes of 3×P3-DsRed and FL-HSA was constructed ( image 3 ), SV40 Poly A (such as SEQ ID NO.12) and Bombyx mori silk fibroin light chain (FL) Poly A (base such as SEQ ID NO.13, peptide such as SEQ ID NO.1) are the ends of the two expression boxes respectively Signal; the 3×P3 promoter in this plasmid starts red fluorescent protein (DsRed) (gene such as SEQ ID NO.10, amino acid such as SEQ ID NO.11) specifically expresses red fluorescence in the eye; silkworm silk fibroin light chain promoter starts Human serum albumin (HSA) (eg, SEQ ID NO.15) is specifically expressed in the posterior silk gland of the silkworm, and the silk fibroin light chain signal peptide can promote the secretion of human serum albumin from the cells into the lumen of the silk gland of the silkworm.

[0033] The novel helper pl...

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Abstract

The invention discloses an efficient transgenosis method with mediation of a transcription activator-like effector (TALE) protein. The method comprises the steps that a helper plasmid containing a transcription activator-like effector protein DNA (Deoxyribonucleic Acid) sequence and piggyBac transposon (PBase) DNA sequence is constructed by adopting a molecular biological technique, and transcribed to mRNA (Messenger Ribonucleic Acid) in vitro; mRNA of the helper plasmid and DNA of a piggyBac transposon plasmid containing an exogenous gene are imported to a fertilized egg of an animal by adopting a microinjection method; a transgenic animal of which the exogenous gene can be stably inherited and expresses is obtained; and a transgenosis positive individual is screened out according to a marker gene or the expression characteristic of the gene. With the adoption of the method, a piggyBac transposon can be efficiently inserted into a genome of a host organism by the action of a TALE-PBase fusion protein; the transgenosis efficiency mediated by the piggyBac transposon can be improved remarkably; and helps are offered to researches such as transgenic animal acquirement and insertion mutagenesis.

Description

technical field [0001] The invention relates to a transgenic method, in particular to a transgenic transgenic method mediated by a transcriptional activator-like effector protein in the field of transgenic organisms. Background technique [0002] Transcriptional activator-like effector protein (TALE) is a conserved bacterial effector protein isolated from the pathogenic genus Xanthomonas spp. It is a kind of protein that can specifically recognize DNA sequences. So far, TALE has been widely used in gene targeting, site-specific regulation of endogenous genes and site-specific epigenetic modification. [0003] In the past ten years, piggyBac transposon-mediated transgenic technology has been successfully applied to a large number of vertebrates and invertebrates, and has become a powerful genetic manipulation tool. important role. However, the transgenic efficiency mediated by piggyBac transposons is still low, and the transformation efficiency is unstable. In recent years,...

Claims

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Application Information

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IPC IPC(8): C12N15/89C12N15/85C12N15/62C12N15/54A01K67/04
Inventor 钟伯雄叶露鹏钱秋杰尤征英车家倩张玉玉宋佳
Owner ZHEJIANG UNIV
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