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Method and kit for down-regulating eukaryotic gene expression

A gene expression and eukaryotic technology, applied in the field of genetic engineering, can solve the problems of unstable down-regulated gene efficiency and short maintenance time of down-regulated genes, and achieve the effect of high specificity and good stability

Active Publication Date: 2017-04-19
NANTONG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the application of Morpholino, like many gene downregulation methods, has many limitations
The way Morpholino is down-regulated is not through stable genetic manipulation, so the efficiency of its down-regulated genes is very unstable, and the maintenance time of its down-regulated genes is also short

Method used

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  • Method and kit for down-regulating eukaryotic gene expression
  • Method and kit for down-regulating eukaryotic gene expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Down-regulation of fabp11a gene using 5'-p-ss-DNA Oligo / NgAgo system:

[0024] 1. Transform and synthesize NgAgo gene: introduce one or two nuclear localization sequences at the end of the NgAgo gene sequence, add polyA at the 3' end, add 5'-UTR sequence and promoter SP6 / T7 / T3 sequence at the 5' end . The modified NgAgo gene sequence is packaged with the pCS2+ vector: the modified NgAgo gene sequence and the pCS2+ vector are double digested to obtain the DNA sequence of the cohesive end, the DNA sequence is purified and recovered, and the DNA sequence of the cohesive end is catalyzed by ligase, purified and recovered to obtain the expression of pCS2+-NgAgo Plasmid (SEQ. ID. NO. 3). The pCS2+-NgAgo plasmid was linearized, in vitro transcribed, and recovered to obtain Argonautem RNA. For details, refer to the experimental procedure of a commercial in vitro transcription kit.

[0025] 2. According to the fabp11a genomic DNA sequence information of the ensembl database

...

Embodiment 2

[0030] Down-regulation of ntl gene by 5'-p-ss-DNA Oligo / NgAgo system

[0031] Steps are the same as Example 1

[0032] 2. According to the ntl genome DNA sequence information of the ensembl database

[0033] (http: / / asia.ensembl.org / Danio_rerio / Gene / Summary?db=core; g=ENSDARG00000017299), design and synthesis of 5'-p-ss-DNA Oligo, such as 5'-p-ss-DNA Oligo ntl -f (SEQ.ID.NO.6), 5'-p-ss-DNA Oligo ntl-r (SEQ.ID.NO.7).

[0034] 3. Argonaute mRNA and 5'-p-ss-DNA Oligo ntl-f or ntl-r were co-injected into zebrafish 1-cell stage fertilized eggs. The microinjection amount of each fertilized egg was controlled in the range of 0.2-0.5ng of NgAgo mRNA and 0.06-0.15nM of 5'-p-ss-DNAOligo.

[0035] 4. When the injected embryo develops to 24-72hpf, observe its developmental shape. Use a genomic DNA extraction kit to extract genomic DNA from zebrafish embryos: place the embryos in cell lysate and incubate at 65°C for 30 minutes, at 95°C for 5 minutes, and at 16°C for 1 minute, (refer to...

Embodiment 3

[0037] Down-regulation of fabp11a gene using 5'-p-ss-DNA Oligo / NgAgo system:

[0038] 1. Transform and synthesize NgAgo gene: introduce one or two nuclear localization sequences at the end of the NgAgo gene sequence, add polyA at the 3' end, add 5'-UTR sequence and promoter SP6 / T7 / T3 sequence at the 5' end . The modified NgAgo gene sequence is packaged with the pcDNA3.1(+) vector: the modified NgAgo gene sequence and the pcDNA3.1(+) vector are double-digested to obtain the DNA sequence of the cohesive end, the DNA sequence is purified and recovered, and the ligase catalyzes the cohesive end The DNA sequence was purified and recovered to obtain the pcDNA3.1(+)-NgAgo expression plasmid (SEQ.ID.NO.8). The pcDNA3.1(+)-NgAgo plasmid was linearized, in vitro transcribed, and recovered to obtain Argonaute mRNA. For details, refer to the experimental procedure of a commercial in vitro transcription kit.

[0039] Step 2 to step 4 are the same as in Example 1.

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Abstract

The invention discloses a method and kit for down-regulating the eukaryotic gene expression. The gene expression is down-regulated through combining 5'-phosphorylated single-stranded oligodeoxynucleotide (5'-p-ss-DNA Oligo) and Argonaute mRNA. The method comprises the following steps: reconstructing NgAgo gene, constructing an expression plasmid, carrying out linearization, in vitro transcription and recovery on the expression plasmid to obtain the Argonaute mRNA, designing and synthesizing the 5'-p-ss-DNA Oligo according to a target gene sequence, and introducing the Argonaute mRNA and the 5'-p-ss-DNA Oligo to a target cell. The NgAgo gene constructed in the method can rapidly and highly-efficiently enter a nucleus in the target cell; the 5'-p-ss-DNA Oligo sequence designed according to the target gene has good specificity on target sequence identification; a 5'-p-ss-DNA Oligo / NgAgo system can stably and highly-efficiently down-regulate the target gene expression through the inhibition effect on an eukaryotic cell in the DNA level; the 5'-p-ss-DNA Oligo has good stability, and well plays a role in the cells; and no endogenous 5'-p-ss-DNA Oligo exists in the eukaryotic cell under natural conditions, so the 5'-p-ss-DNA Oligo has high specificity.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a method for down-regulating eukaryotic gene expression and a kit thereof. Background technique [0002] Gene expression down-regulation technology refers to the genetic manipulation technology that inhibits the transcription and mRNA translation of the target gene at the molecular level to reduce the expression of the target gene. The principle of this technology is to inhibit the transcription of the target gene, inactivate post-transcriptional cleavage, and reduce the translation of mRNA through genetic modification or introduction of short strands of DNA or RNA (complementary to the target gene or mRNA transcript). Expression is downregulated. Currently, gene expression down-regulation technologies widely used include RNA interference (RNAi) technology and Morpholino technology. [0003] RNAi technology is a sequence-specific gene downregulation techn...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/89C12N15/66C12N15/113
CPCC12N15/113C12N15/66C12N15/89C12N2310/10
Inventor 刘东石运伟董张及王新
Owner NANTONG UNIVERSITY
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