Genetic modification method of target gene in genome

A genetic transformation and genome technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, and the use of vectors to introduce foreign genetic materials, can solve the problem of low probability of success

Active Publication Date: 2015-04-15
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] On the other hand, in the prior art, the success probability of genetic modification of multiple sites of the target genome is very low, so those skilled in the art are committed to developing a technology that can improve the success rate of multi-site genetic modification

Method used

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  • Genetic modification method of target gene in genome
  • Genetic modification method of target gene in genome
  • Genetic modification method of target gene in genome

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0185] Embodiment 1, construction Red recombinase+I-SceI endonuclease bifunctional auxiliary (helper) plasmid

[0186] 1. Construction of plasmids expressing I-SceI with rha promoter and λRED recombinase with arabinose promoter

[0187] The specific operation steps are: rhaRas (NcoI) and rhaov primers amplify the genome of Escherichia coli w3110 to obtain Fragment B (about 2 kb) containing a rhamnose promoter (rhaB promoter). I-SceIup and I-SceIdn (NcoI) amplify the pUC19RP12 plasmid (Nucl. Acids Res. (1999) 27 (22): 4409-4415) to obtain fragment C (about 0.7kb) containing the I-SceI endonuclease gene .

[0188] rhaRas (NcoI): ACCATGGGGCATGGCGAATTAATCT (SEQ ID NO: 1);

[0189] I-SceIdn (NcoI): TCCATGGTTATTATTTCAGGAAA (SEQ ID NO: 2);

[0190] I-SceIup: ATGCATCAAAAAAACC (SEQ ID NO: 3);

[0191] Rhaov: GGTTCATTACCTGGTTTTTTTTGATGCATAATGTGATCCTGCTGAAT (SEQ ID NO: 4).

[0192] The two fragments B and C were linked together by Overlap PCR and cloned into pMD18simple-T vector. T...

Embodiment 2

[0215] Embodiment 2, construction has the cloning vector of I-SceI restriction site

[0216]First, a 1.5 kb fragment containing the kanMX gene was excised from pUG6 (from EUROSCARF; see Güldener, U. et al.; 1996; Nucleic Acids Research24, 2519-2524) with NotI, and connected into pBluecript II KS(-) (Nucleic Acids Res17 :9494, from the NotI site of Stratagene) to obtain pKS-K.

[0217] The kanMX gene of pKS-K was amplified with primers T3_IsceI and T7_IsceI, digested with BssHII, and reconnected into the BssHII site of pBluecriptIIKS(-). According to the connection direction, two plasmids, pKSKI-1 (the direction of the kanMX gene and the direction of the lacZ fragment of pBluecriptIIKS(-) itself are opposite) and pKSKI-2 (the direction of the kanMX gene and the direction of the lacZ fragment of pBluecriptIIKS(-) itself are the same) were obtained respectively.

[0218] T3_IsceI: AAGCGCGC CCAATTAACCCTCACTAA (SEQ ID NO: 13);

[0219] T7_IsceI: CAGCGCGC TGTAATACGACTCACTA...

Embodiment 3

[0227] Embodiment 3, construction is used for knocking in the template plasmid

[0228] Using pIJ773 and pIJ778 plasmids as templates (PNAS2003100 (4) 1541-1546) respectively, the primer pair Apr-ISceI-F and Apr-ISceI-R were amplified to obtain 1.5 kb containing apramycin (Apr) or spectinomycin ( Spc) resistant fragments. After adding A, it was connected into pMD18-T simple (TAKARA) to obtain pMD-ISceI-apr-ISceI and pMD-ISceI-spc-ISceI plasmids. Such as image 3 and Figure 4 .

[0229] Apr-ISceI-F: 5'TCCCCCGGGGCTAGGGATAACAGGGTAATATTCCGGGGATCCGTCGACC3' (SEQ ID NO: 15);

[0230] Apr-ISceI-R: 5'TCCCCCGGGGCATTACCCTGTTATCCCTAGTGTAGGCTGGAGCTGCTTC3' (SEQ ID NO: 16).

[0231] The pKD3 plasmid (PNAS2003100(4)1541-1546) was digested with XbaI to obtain a 1.0 kb fragment containing chloramphenicol (Cm) resistance. At the same time, the pMD-ISceI-apr-ISceI plasmid was also digested with XbaI, the apramycin resistance fragment was excised, and the vector backbone was retained. Conn...

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Abstract

The invention relates to a genetic modification method of a target gene in a genome, and provides a new genetic modification (such as knockout, site directed mutagenesis and / or gene knock-in) method of the target gene in the genome. The method has the advantages of simple operation steps, easy enforcement, high modification accuracy, high efficiency, and no superfluous sequence residual in the genome after the modification.

Description

technical field [0001] The invention belongs to the field of gene technology; more specifically, the invention relates to a method for genetic modification of a target gene in a genome. Background technique [0002] Biological research in the post-genome era urgently needs an effective gene function analysis method. The application of genetic modification provides strong support for studying the function of genes and finding new interventions for treating diseases. The genetic modification of the genome mainly involves gene knockout, knockin, substitution, point mutation, etc. http: / / www.intechopen.com / books / genetic-manipulation-of-dna-and-protein-examp les-from-current-research / site-directed-mutagenesis-using-oligonucleotide-based-recombining , a series of methods for modifying specific loci (genes) on the Escherichia coli genome were reviewed. [0003] At present, the main method of Escherichia coli genome modification is the method based on Red recombination. Knockout...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N1/21C12R1/19
Inventor 杨俊杰孙兵兵黄鹤杨晟
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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