Difference between revisions of "Team:NJU-China/Notebook"

Team member recruitment

First meet up, laboratory safety regulation learning and task assignment.

Learn experimental techniques like western blot, RT-PCR, cell culture techniques, etc.
Data collection and project discussion
Collaboration with SYSU-Software

Human practice：Interview with doctors and patients from Nanjing General Hospital and project design.

Model built

Anti-Bcl2 plasmid, tpep plasmid design and construction

Targeting verification in vitro
HEK293 cell was stained with fluorescence and Tpep plasmid was transfected, two days later, the cells was collected and exosome was extracted. Then the exosomes were co-cultured with C2C12 cell and mouse adipose cell 3T3L1 for 12 hours individually, cell nucleus were dyed with DAPI, then the cells were observed under fluorescent microscope.

Exosome characterization
TEM and NTA experiments were carried out in the assay of exosomes.
Endotoxin test for exosomes

Anti-Bcl2 and Tpep plasmid were co-transfected into HEK 293 cell, then the exosome extracted was co-cultured with adipose cell. Protein and RNA were extracted and stored for further research.

Run three times western blot to detect whether there is down-regulated expression of BCL2 protein in adipose cell after plasmids transfection, all failed.

Bcl2 RNA reverse transcription and Qpcr to exam the expression level of BCL2 in experimental group, Bcl2 RNA expression marginally reduced.

Bcl2 plasmid redesign and improvement, repeat experiments to value the effect of the new plasmid.
Interlab
Plasmids transformation into E.coli.
Pick single colonies and run colony PCR.
OD600 measurement , plasmid extraction and DNA electrophoresis.
Measure GFP fluorescence intensity with microplate reader
Flow cytometry

Egfr tracer experiment in vivo
4 mice was divided into two groups, experimental group was tail intravenously injected with egfr siRNA+tpep exosome, while control group egfr siRNA exosome. 12hours later, the mice was dissected, their heart, liver, spleen, lung, kidney and adipose tissue were separated.
RNA extraction of those tissue, reverse transcription and Qpcr was executed to compare egfr expression level in different tissue.

Aug 15 – Sep 5 Valuation in vivo
Aug 15- Aug20 Plasmid maxi extraction, cell transfection and exosome extraction.
Aug21 –Sep 5 20 mice were fat- fed and divided into fore groups, 5 in each group. Group I was intravenously injected with PBS every two days, Group II egfr-tpep exosome , group III and Group IV Bcl2-tepe exosome, Weight data was collected every day, no obvious tendency in weight lose. After two weeks, took blood by removing eyeball, and heart liver spleen lung kidney and adipose tissue were separated, adipose tissue was weighted.
ITT
Starvation treatment in group IV for 6 hours, intraperitoneally inject insulin 0.75 unit per kg, Took blood sample form tail end and measure blood glucose at the time of 0, 15, 30, 45, 60, 90, 120 min.
GTT
Starvation treatment overnight and intraperitoneally inject glucose, the following step is as same as ITT

LDL, HDL,CHO, TG measurement of blood serum, failed
protein and RNA extraction of liver and adipose, RNA reverse transcription.

Western blot of liver and adipose protein to exam Bcl2 expression level, the protein was not so pure that it showed obscure strip
Repeated experiment: Centrifugate the protein before loading, obscure strip disappeared, still ,no tendency.
Sep 14 Sent fat tissue to be made into sections, stained by means of hematoxylin eosin (HE) method and calculated under the Image Analyzer.
Sep 9 Bcl2-mRNA Qpcr with the method of SYBR Green

Repeated exprement: Validation in vivo, exosomes were injected every two days and weight data was collected everyday. Four weeks later, took blood by removing eyeball, and heart liver spleen lung kidney and adipose tissue were separated. ITT and GTT were also carried out. Western blot and Qpcr were used to exam Bcl2 protein and Bcl2 mRNA individually, failed.
Oct 9 Part submission to Nanjing Genescript

Final Data analysis and preparation.