Method for constructing doxycycline-regulated rat bone marrow mesenchymal stem cell line expressing c-met protein
A bone marrow mesenchymal, expression regulation technology, applied in the field of cell biology, can solve problems such as unseen, lack of control of expression sites or cell types, etc.
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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 (1μg / μL)
2
...
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
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