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Model mouse preparation method and recombinant vector for conditional cell removal

A recombinant vector and model mouse technology, applied in the direction of recombinant DNA technology, the introduction of foreign genetic material using a vector, and the cells modified by the introduction of foreign genetic material, etc. Scientific research and practical applications, etc., to achieve accurate and intuitive experimental results and shorten the time.

Active Publication Date: 2013-11-27
BIOCYTOGEN PHARMACEUTICALS (BEIJING) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The defects of these two models of mice are: all the cells expressing Cre are killed, the fluorescent protein reporter gene cannot be used to track whether the cells expressing Cre have been knocked out from the body, and the expression of the target gene cannot be time-wise. or lack of control
The disadvantage of this method is that: generally more than 10 generations of backcrossing are required to obtain homozygous model mice, and backcrossing often takes 2 years, which requires a lot of research effort and capital investment; on the other hand, because backcrossing Different times will also cause poor repeatability of experimental results, which is not conducive to scientific research and practical application
This is also a big flaw for the development and production of standard model animals

Method used

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  • Model mouse preparation method and recombinant vector for conditional cell removal
  • Model mouse preparation method and recombinant vector for conditional cell removal
  • Model mouse preparation method and recombinant vector for conditional cell removal

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preparation example Construction

[0043] The invention also provides a preparation method of the recombinant vector, which comprises introducing the DNA fragment structure iDTR-2A-FP into the expression vector Ai3Rosa26.

[0044] Specifically, the construction method of the recombinant vector can be as follows: design primers according to the multiple cloning sites in iDTR, 2A, FP and expression vectors, amplify by PCR method, and then connect iDTR, 2A and FP by PCR method , purify and recover the DNA fragment structure iDTR-2A-FP obtained by ligation, and then insert the DNA fragment structure iDTR-2A-FP into the multiple cloning site in the expression vector by double enzyme digestion method to obtain a recombinant vector.

[0045] In the present invention, the reagents used in the PCR reaction may be PCR reagents routinely used in the art.

[0046] In the present invention, the PCR reaction product can be purified and recovered by using a DNA fragment recovery method commonly used in the art, such as a DNA ...

Embodiment 1

[0069] This example is used to illustrate the preparation method of the recombinant vector provided by the present invention.

[0070] 1. Construction of recombinant vector

[0071] 1. Amplify the coding gene iDTR of diphtheria toxin receptor, the base sequence shown in SEQ ID NO: 2 of coding polypeptide fragment 2A and the coding gene EGFP of fluorescent protein by PCR reaction respectively.

[0072] (1) Primers:

[0073] DTR-F: 5'-cgatggccggccaccatgaagctgctgccgt-3'

[0074] DTR-R1(in): 5'-gtcaaaattcaaagtctgtttcaccgggtgggaattagtc atgcccaact-3'

[0075] DTR-F1(in): 5'-agttgggcatgactaattcccacccggtgaaacagactttgaattttgac-3'

[0076] DTR-R2 (in): 5'-tcctcgcccttgctcaccatgggccctgggttggactc-3'

[0077] DTR-F2(in): 5'-gagtccaacccagggcccatggtgagcaagggcgagga-3'

[0078] DTR-R(in): 5'-cgatggccggccacttacttgtacagctcgtccatgc-3'

[0079] in,

[0080] The primer pair DTR-F and DTR-R1(in) are used to amplify the coding gene of diphtheria toxin receptor;

[0081] The primer pair DTR-F1(in...

Embodiment 2

[0096] This example is used to illustrate the preparation method of the cell knockout model mouse provided by the present invention:

[0097] (1) Culture, transfection and positive clone screening of embryonic stem cells:

[0098] 1. Culture of embryonic stem cells

[0099] Culture C57BL / 6 embryonic stem cells in a culture dish lined with feeder cells, and place at 37°C, 5% CO 2 , saturated humidity incubator culture. The composition of the medium used is shown in Table 1:

[0100] Table 1

[0101]

[0102]

[0103] 2. Electroporation transfection

[0104] Confirm that the growth of C57BL / 6 embryonic stem cells is in good condition before transfection.

[0105] Remove the 100mm Petri dish full of cells from CO 2 After being taken out of the incubator, the stem cell culture medium in the 100mm plate was sucked away. Add 5ml of PBS along the wall of each dish, shake it gently, suck it out, and wash it twice. Add 1.5ml of 0.25% trypsin along the wall of each dish, s...

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Abstract

The invention discloses a recombinant vector for conditional cell removal and a preparation method thereof. The recombinant vector comprises a DNA (Deoxyribonucleic Acid) fragment structure iDTR-2A-FP, wherein iDTR is a coding gene of a diphtheria toxin receptor, 2A can realize a coded nucleic acid sequence of a sheared polypeptide fragment through ribosome jumping, and FP is a coding gene of fluorescent protein. The invention further provides a method for preparing a model mouse for conditional cell removal. A descendant mouse, obtained after the model mouse for cell removal, prepared by the method provided by the invention, is mated with a Cre mouse, has the following characteristics: through expression conditions of the fluorescent protein, direct detection on cell removal efficiency can be carried out, and whether cells are completely removed from in vivo or not can be determined, so that more accurate and intuitive results can be obtained; and the expression or deletion of target genes can be controlled from time through controlling the inducer giving time.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant vector for conditional cell elimination, a preparation method thereof, and a method for establishing a conditional cell elimination model mouse Background technique [0002] Conditional cell deletion is based on the chromosomal site-specific recombinase system, and the commonly used site-specific recombinase systems are Cre-LoxP and Flp-FRT systems. For example, a pair of LoxP-Stop-LoxP sequences is placed upstream of the DNA sequence of a conditional lethal gene to be knocked in to obtain a conditional cell knockout model mouse. Then, the conditional cell knockout model mice were bred with the Cre mice with cell-specific expression to obtain mice with conditional lethal gene knock-in in specific cells, that is, to obtain conditional gene knock-in mice. Then, the specific cells can be knocked out by controlling the administration time of the inducer. [0003] Diphther...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/66C12N15/65C12N5/10A01K67/027
Inventor 沈月雷
Owner BIOCYTOGEN PHARMACEUTICALS (BEIJING) CO LTD
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