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Gene knock-in non-human animal

A non-human animal and genetic technology, applied in the direction of anti-animal/human immunoglobulin, drug combination, biological testing, etc., can solve the problem of weak effect

Active Publication Date: 2015-07-22
CHUGAI PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Another recently reported paper states that NMD is suppressed if the hp7 sequence is present between the PTC that is the target of NMD and the exon-exon junction (Non-Patent Document 4), but the effect is weak

Method used

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  • Gene knock-in non-human animal
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  • Gene knock-in non-human animal

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] [Example 1] Preparation of human IL6R gene knock-in mice

[0105] (1) Construction of knock-in vector

[0106] The E. coli artificial chromosome (BAC) cloned with the genomic region of the mouse interleukin-6 gene (I16ra) was used. The target region of the mouse Il6ra gene on the BAC uses the Red / ET system (GeneBridges) to insert the coding sequence of the human interleukin-6 receptor gene (GeneBank#NM000565), hp7 sequence, and poly A addition signal through homologous recombination , LoxP sequence, neomycin resistance (neo) gene cassette and loxP are connected in sequence. At this time, insert the translation initiation site of exon 1 of the mouse Il6ra gene on the BAC so that it coincides with the translation initiation site of the human IL6R gene, and make the exon of the mouse Il6ra gene 1 The base sequence defect after the translation start site inside is equivalent to 40 base pairs. In addition, the pgk gene promoter is added to neo, which is a drug resistance gene,...

Embodiment 2

[0128] [Example 2] Establishment and evaluation of humanized giant lymph node hyperplasia model mice

[0129] (1) Establishment of humanized giant lymph node hyperplasia model mice

[0130] The hIL6R knock-in mice were bred with H-2Ld human IL6 transgenic mice (Cytokine. 2002, Dec. 21; 20(6): 304-311) to make double transgenic mice. The genotype was analyzed by PCR using genomic DNA extracted from the tissue of each individual, and individuals with a homozygous hIL6R knock-in array and hIL6 transgene were selected. The detection of hIL6R knock-in array uses the above-mentioned PCR reaction system. That is, the PCR reaction solution consisted of 1 μl of sample, 12.5 μl of 2×GC buffer I, 4 μl of dNTP (2.5 mM), 0.25 μl of primers (50 μM each), 0.25 μl of LA Taq (TAKARA), and 6.75 μl of distilled water (25 μl in total) ). In addition, PCR conditions were a preheating at 94°C for 4 minutes, 35 cycles of amplification cycles at 94°C for 30 seconds, 62°C for 30 seconds, and 72°C for 3 ...

Embodiment 3

[0146] [Example 3] Knock-in of neomycin resistance gene into mouse Pou5f1 locus

[0147] (1) Construction of knock-in vector

[0148] The neomycin resistance (neo) gene was inserted so that the translation initiation site of exon 1 of the mouse Pou5f1 gene coincides with the translation initiation site of the neomycin resistance (neo) gene, Furthermore, the base sequence defect after the translation initiation site inside exon 1 of the mouse Pou5f1 gene was equivalent to 107 base pairs to construct a knock-in vector. At this time, construct the hp7pA vector with the hp7 sequence and the poly A addition signal immediately below the stop codon of the neo gene, and the hp7 pA vector without the hp7 sequence and the poly A addition signal, but the endogenous poly A addition signal The control vector. In addition, as the homology arm of the knock-in vector, a structure of 1.7 kb upstream of the genomic DNA reaching the translation initiation site of the Pou5f1 gene and about 5 kb geno...

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Abstract

The present invention provides a non-human animal in which a DNA comprising an hp7 sequence-encoding DNA and a poly A addition signal-encoding DNA added on the 3' side of a DNA encoding an arbitrary foreign gene is inserted in the same reading frame as that of an arbitrary target gene present on the genome of the non-human animal.

Description

Technical field [0001] The present invention relates to a method for evaluating gene knock-in non-human animals and compounds using gene knock-in non-human animals. Background technique [0002] Therapeutic drugs with high specificity to molecular targets, such as antibody drugs, have been developed in large numbers. In order to perform more accurate clinical evaluations, the demand for humanized non-human animals such as humanized mice has increased. As methods for making humanized mice, so-called transgenic mice in which mice express human genes, and gene targeting methods in which mouse genes are replaced with human genes are widely known. Humanized mice made by the above methods are widely used. report. However, in mice such as transgenic mice that are simply introduced with vectors for expressing human genes, most of them do not exhibit the expected expression pattern. For example, a mouse obtained by overexpressing the human interleukin-6 (IL-6) receptor gene using the pC...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/027C12P21/08G01N33/15G01N33/50G01N33/68C07K16/28C07K16/46C12N15/09
CPCG01N33/5088G01N2800/24G01N33/15A01K67/027C12N15/09G01N33/6869A01K67/0278A01K2227/105A01K2217/072A01K2267/0387C07K14/7155A61P29/00A61P7/00A61K49/0008C07K16/2866C07K2317/24G01N33/6854G01N2333/7155
Inventor 寺社下浩一上田乙也
Owner CHUGAI PHARMA CO LTD
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