Team:NJU-China/Notebook/Calendar
Week 1 (05/22/2016-05/28/2016)
Learn laboratory safety regulations and lab skills like PCR, restriction enzyme digestion, electrophoresis, fragment ligation, etc.
Acquire iRGD-Lamp2b fusion protein sequence from GenScript, then try to construct plasmid (pcDNA6.2) ---Failed
Week 2 (05/29/2016-06/04/2016)
PCR, Plasmid construction using iRGD-Lamp2b sequence and pSB1C3 backbone.---Failed
Plasmid construction using iRGD-Lamp2b sequence and pSB1C3 backbone.---Failed
Learn cellular experiment skills. (cell culture & transfection)
HEK293, A549-Luc, A549 cell lines recovery.
Week 3 (06/05/2016-06/11/2016)
Plasmid construction using iRGD-Lamp2b sequence and pSB1C3 backbone.---Succeed
Learn western blot protocols.
Week 4 (06/12/2016-06/18/2016)
Test software written by team SYSU-Software . Design siRNAs.
Order siRNA from GenScript.
Week 5 (06/19/2016-06/25/2016)
Receive siRNA from GenScript, Test RNA interference efficiency by cell transfection and western blotting.--- Failed
Test KRAS expression level in which cell line using transfection and western blotting.---Succeed
Abandon cells contaminated with bacteria. Disinfection and sterilization in cell incubators.
Learn protocols for FCFM, ultra-high-speed centrifuge, in-vivo imaging.
Obtain access for State Key Laboratory of Pharmaceutical Biotechnology.
Week 6 (06/26/2016-07/02/2016)
A549-Luc cell line recovery and culture. Preparation for animal modeling.
Design shRNA based on validated siRNA sequence. Send our designed sequence to GenScript for synthesis.
Successfully loading our exosomes with iRGD and siRNA for the first time. Send exosome samples to National University of Defense Technology for electron microscopy assay and Nanosight.
Week 7 (07/03/2016-07/09/2016)
One-week suspension for thoroughly disinfection and sterilization.
Week 8 (07/10/2016-07/16/2016)
Receive feedbacks from National University of Defense Technology.
HEK293, A549-Luc, A549 cell lines recovery.
Detecting KRAS expression level using western blotting and qPCR.
A549-Luc cell proliferation for mouse tumor model construction.
Verify the effect of exosome on cell proliferation of A549
Week 9 (07/17/2016-07/23/2016)
A549-Luc cell proliferation in preparation for tumor implantation
HEK293 cell proliferation for mass-production of exosomes
Order nude mice from Model Animal Research Center of Nanjing University and inoculate A549-Luc subcutaneously into nude mice to develop implant-tumor.
Receive two single strands of RNA containing KRAS shRNA sequence from Genscript
Learn work standards of animal experiments. Obtain permission for animal experiment in School of Life Science, NJU
Week 10 (07/24/2016-07/30/2016)
Abandon HEK293 cells with obviously slower cell proliferation and changing characteristics. Recover HEK293 cells.
Anneal two single strands of shRNA into segments. Try to insert these fragments into pSB1C3 plasmid vectors. ---Failed
Week 11 (07/31/2016-08/06/2016)
Detect endotoxin level of exosome
HEK293 cell proliferation
Retry to anneal two single strands of shRNA into segments and insert them into pSB1C3 plasmid vectors. ---succeed
Week 12 (08/07/2016-08/13/2016)
Perform in vivo imaging on nude mice for experimental model and most tumor implantations were successful
Randomly divide the mouse model into two groups
HEK293 cell proliferation
HEK293 cells amplification rates decrease and cells are thawed
Week 13 (08/14/2016-08/20/2016)
HEK293 cell proliferation
HEK293 cell are abandoned for decreased viability.
Week 14 (08/21/2016-08/27/2016)
HEK293 cell line recovery and proliferation.
Week 15 (08/28/2016-09/03/2016)
HEK293 cell proliferation
Week16 (09/04/2016-09/10/2016)
Obtain enough HEK293 cells. Transfect HEK293 cells with KRAS siRNA. Isolate exosomes from cell culture.
Mouse tail vein injection
Week17 (09/11/2016-09/17/2016)
HEK 293 cell proliferation. Transfect these cells and collect exosome
Mouse tail vein injection
Week18 (09/18/2016-09/24/2016)
Stop mouse tail vein injection ahead of schedule since time was limited
Detect the effects of our exosome through in vivo imaging
Sacrifice these nude mice, collect their tumor tissues and record data
Week19 (09/25/2016-10/01/2016)
Detect the expression level of KRAS mRNA and K-ras protein in these tumor tissues
Frozen sectioning using tumor tissues. Observe relevant pathological characteristics.
