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Gene knock-in recombinant vector and preparation method thereof as well as method for preparing mouse model

A recombinant vector and gene knock-in technology, applied in the biological field, can solve the problems of inconvenient and flexible operation, high requirements for experimental instruments and equipment, and low fluorescence sensitivity, and achieve the effects of clean background, accurate experimental results, and high detection sensitivity

Active Publication Date: 2014-03-19
BIOCYTOGEN PHARMACEUTICALS (BEIJING) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This kind of model can only be detected by a small animal in vivo imager that can detect green fluorescence or by extracting tissues and cells after killing mice and observing them with a fluorescence microscope. It has high requirements for experimental instruments and equipment, and the operation is not convenient and flexible. low sensitivity

Method used

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  • Gene knock-in recombinant vector and preparation method thereof as well as method for preparing mouse model
  • Gene knock-in recombinant vector and preparation method thereof as well as method for preparing mouse model
  • Gene knock-in recombinant vector and preparation method thereof as well as method for preparing mouse model

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preparation example Construction

[0033] The present invention also provides a method for preparing the recombinant vector, wherein the method includes introducing the DNA fragment structure IRES-luc into the expression vector pIL17A-GFP. For example, primers can be designed according to IRES, luc and the cloning site in the vector, the IRES and luc sequences can be obtained by PCR, the IRES and luc sequences can be purified and recovered, and the DNA fragment structure IRES-luc can be obtained by PCR connection, and then double enzyme digestion method can be used to The DNA fragment structure IRES-luc was inserted between the cloning sites XhoI and XmaI in the expression vector to obtain a recombinant vector.

[0034] In a specific embodiment of the present invention, the preparation method may include the following steps: design primers according to IRES, Gluc and the cloning site in the vector, and obtain IRES and Gluc sequence, purification and recovery of IRES and Gluc sequences and PCR connection to obta...

Embodiment 1

[0066] This example is used to illustrate the construction method of the gene knock-in vector provided by the present invention.

[0067] 1. Amplify the coding sequence of IRES and the coding sequence of luciferase Gluc by PCR reaction respectively, and the primers used are shown in Table 1.

[0068] (1) Primers:

[0069] Table 1

[0070] Primer name

nucleic acid sequence

IRES-F

5'-atctctcgaggttaacgaattccgccccccccccctaacgtta-3'

IRES-R

5'-gaactttgactcccatggttgtggccatattatcatcgtgtttttcaaag-3'

Gluc-F

5'-ctttgaaaaacacgatgataatatggccacaaccatgggagtcaaagttc-3'

Gluc-R

5'-acatcccgggcttagtcaccacc-3'

[0071] in,

[0072] The primer pair IRES-F and IRES-R are used to amplify the coding nucleotide sequence of the IRES gene from the pT7CFE1 plasmid;

[0073] The primer pair Gluc-F and Gluc-R was used to amplify the coding nucleotide sequence of the Gluc gene from the pMCS-Gluc plasmid.

[0074] The primer pair IRES-F and G...

Embodiment 2

[0090] This embodiment is used to illustrate the preparation method of the mouse model provided by the present invention:

[0091] (1) Culture, transfection and positive clone screening of embryonic stem cells:

[0092] 1. Culture of embryonic stem cells

[0093] C57BL / 6 embryonic stem cells were cultured in a culture dish covered with feeder cells, and placed in an incubator at 37°C, 5% CO2, and saturated humidity. The composition of the medium used is as follows in Table 2:

[0094] Table 2

[0095] Medium composition

volume

Knockout DMEM

500ml

FBS

90ml

MEM NEAA

6ml

L-Glutamine

6ml

ESGRO LiF

60μL

β-mercaptoethanol

600μL

[0096] 2. Electroporation transfection

[0097] After the 100mm culture dish full of cells is taken out from the CO2 incubator, the stem cell culture medium of the 100mm dish is aspirated. Add 5ml of PBS along the wall of each dish, shake it gently, suck it out, and w...

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Abstract

The invention discloses a recombinant vector for gene knock-in. The recombinant vector comprises a DNA (deoxyribonucleic acid) fragment structure IL17A-IRES-luc, wherein IL17A is an encoding gene of interleukin 17A, IRES is internal ribosome entry site, and luc is an encoding gene of secreting luciferase. The invention also provides a method for preparing the gene knock-in recombinant vector and a mouse model prepared by using the recombinant vector, wherein the mouse model can be used for detecting the expression of the IL17A gene by detecting the expression of the secreting luciferase in blood or urine; a fluorescent microscope or other complicated equipment is not needed; the secreting luciferase emits light by virtue of an oxidizing reaction of substrate coelenterazine in the catalysis of luciferase, and laser light is not needed, so that the fluorescent background is far lower than GFP (green fluorescent protein) fluorescence, the detection sensitivity is higher, the background is cleaner, and the experiment results are more accurate.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing a mouse model and a gene knock-in recombinant vector. Background technique [0002] Gene knock-in (Knockin) is a technology that introduces specific mutations or exogenous genes at the position of the target gene according to experimental requirements, such as introducing point mutations on the target gene (simulating a human genetic disease model); or introducing a reporter gene (such as EGFP, mRFP , mCherry, etc.) introduce specific sites of the target gene through homologous recombination, so that the expression of the target gene can be tracked and the expression profile of the gene can be studied through the expression of the reporter gene. [0003] IL17A (Interleukin-17A) is a member of the IL-17 family of cytokines and plays a key role in host defense and inflammatory processes. [0004] The current model mouse for monitoring IL17A gene expression is th...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12Q1/66A01K67/027
Inventor 沈月雷
Owner BIOCYTOGEN PHARMACEUTICALS (BEIJING) CO LTD
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