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
View PDF0 Cites 13 Cited by
  • 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

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • 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

Examples

Experimental program
Comparison scheme
Effect test

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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N15/63C12N15/66C12N15/65C12N5/10A01K67/027
Inventor 沈月雷
Owner BIOCYTOGEN PHARMACEUTICALS (BEIJING) CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap