Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Method for constructing rat bone mesenchymal stem cells (BMSCs) cell strain for expressing c-met protein through regulation of doxycycline

A bone marrow mesenchymal, expression-regulating technology, applied in the field of cell biology, can solve the problems of lack of control of the expression site or cell type, and yet to be seen.

Active Publication Date: 2017-03-22
江苏纳华生物科技有限公司
View PDF3 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the controllability of the Tet-on advanced system is mainly reflected in the control of the expression time of the target gene, and the lack of control over the expression site or cell type. Therefore, there are still shortcomings in the field of

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
  • Method for constructing rat bone mesenchymal stem cells (BMSCs) cell strain for expressing c-met protein through regulation of doxycycline
  • Method for constructing rat bone mesenchymal stem cells (BMSCs) cell strain for expressing c-met protein through regulation of doxycycline
  • Method for constructing rat bone mesenchymal stem cells (BMSCs) cell strain for expressing c-met protein through regulation of doxycycline

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1 Extraction and Culture of Rat Bone Marrow Mesenchymal Stem Cells (BMSCs)

[0077] Select 4-week-old SPF grade male SD rats, kill them by cervical dislocation, take out the tibia and femur, remove the metaphysis at both ends, use a 5 ml syringe to draw physiological saline to wash the bone marrow cavity into a 50 ml centrifuge tube, and centrifuge the washing solution at 1000rpm for 10min Finally, use 3ml of 10% serum-containing low-glucose medium to wash and suspend the bottom sediment of the centrifuge tube, add the liquid to a 15ml centrifuge tube containing 3ml of rat lymphocyte separation medium, centrifuge at 2000rpm for 20min, and absorb the third white layer in the centrifuge tube. Membrane layer, and added to a centrifuge tube containing 4 times its volume of normal saline, centrifuged at 1000rpm for 10min, washed with 10% serum low-glucose medium, suspended the bottom pellet and inoculated it into a petri dish, and placed the cells at 37°C with 5% CO ...

Embodiment 2

[0078] Example 2 Establishment of Tet-On Advanced BMSCs cell line

[0079] 1. Construction of lentiviral expression vector GV211-CMV-rtTA

[0080] 1.1 Use SEQ ID NO.1 and SEQ ID NO.2 as upstream and downstream primers respectively, and use the pTet-On advancedVector vector as a template to carry out PCR reaction. The reaction system is shown in Table 1, and the reaction conditions are shown in Table 2; After the mixture was mixed, it was gently blown and mixed, placed in a PCR instrument to amplify the target gene rtTA and the upstream CMV promoter, and recovered with the kit. The correctly sequenced amplification product was the target gene CMV-rtTA.

[0081] Table 1 PCR reaction system

[0082]

[0083]

[0084] Table 2 PCR reaction conditions

[0085]

[0086] Table 3 Enzyme digestion system of lentiviral vector GV211

[0087] Reagent Volume (μl) wxya 2 o

42 10×CutSmart Buffer 5 Lentiviral vector GV211 (1 μg / μL) 2 BamHI, Age ...

Embodiment 3

[0101] Example 3 Establishment of GFP(+) Tet-on-c-met BMSCs cell line

[0102] 1. Construction of lentivirus Len-TRE minCMV-c-met / ALB-GFP

[0103] 1.1 The gene sequence TRE-minCMV promoter-c-met-ALB promoter-GFP was synthesized by Shanghai Jikai Gene Chemical Technology Co., Ltd., its structure is as follows figure 1 As shown, the ALB promoter sequence (237bp) is shown in SEQ ID NO.3, the minCMV promoter sequence (60bp) is shown in SEQ ID NO.4, the TRE sequence (250bp) is shown in SEQ ID NO.5, and the GFP sequence (720bp) as shown in SEQ ID NO.6 ；

[0104] 1.2 Prepare linearized vector DNA and target gene recombination reaction system: 5×CE II Buffer 2 μl, 2.5 μl linearized vector DNA (concentration 800 ng / μL) obtained in step 1.2 of Example 2, TRE-minCMV promoter- c-met-ALB promoter-GFP (concentration 800ng / μL) 1μl, Exnase II 1μl, ddH 2 O supplemented to 10 μl;

[0105] React the above system at 37°C for 30 minutes to obtain the lentiviral expression vector GV211-TRE

...

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

PropertyMeasurementUnit
Virus titeraaaaaaaaaa
Login to View More

Abstract

The invention discloses a method for constructing a rat bone mesenchymal stem cells (BMSCs) cell strain for expressing c-met protein through regulation of doxycycline. The method comprises the steps that an rtTA gene lentiviral expression vector is constructed and subjected to transfection into BMSCs so that a BMSCs cell strain (a Tet-On Advanced BMSCs cell strain) for stably and efficiently expressing rtTA protein can be obtained; then, a Len-TRE min CMV-c-met/ALB-GFP lentiviral vector is constructed and subjected to transfection into the Tet-On Advanced BMSCs, and a flow cytometry is used for screening GFP positive BMSCs; and finally the GFP (+) Tet-on-c-met BMSCs cell strain is obtained. The doxycycline can be added into the obtained BMSCs cell strain to induce the obtained BMSCs to express the c-met protein, and the method has important significance for study on blood doping of the cell strain for treating hepatic failures, the directional homing and differentiation capacities of the BMSCs along with the HGF concentration and the dose-effect relationship between the capacities and the HGF/c-met axis expression level.

Description

technical field [0001] The invention relates to the technical field of cell biology, in particular to a method for constructing a rat bone marrow mesenchymal stem cell strain expressing c-met protein regulated by doxycycline. Background technique [0002] Gene therapy is to transfer exogenous genes with prevention and treatment potential into relevant tissue cells of patients through appropriate vectors, and obtain appropriate expression, so as to achieve the purpose of preventing or alleviating diseases. We have done a lot of work on gene therapy for liver failure, and reviewed the research status and prospects in detail. There are currently two main approaches to gene therapy, namely in vivo and ex vivo. The in vivo approach refers to the assembly of exogenous genes on specific eukaryotic cell expression vectors (viral or non-viral) and direct introduction into the human body. Due to issues such as transfection efficiency and safety, its clinical application range is limi...

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): C12N15/867C12N15/66C12N5/10
CPCC12N5/0663C12N15/66C12N15/86C12N2510/00C12N2740/15043C12N2800/107
Inventor 朱传龙李毓雯王坤李军金柯岳明朱甜甜
Owner 江苏纳华生物科技有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com