Team:BM-AMU/Notebook
MARCH
2019.3.25-2019.3.31
Selection of the experimenters.
APRIL
2019.4.1-2019.4.7
Discussed and decided the project plan and prepared for the experiment equipment and reagents.
2019.4.8-2019.4.21
1. Obtained the sequences of E-Cad and N-Cad, designed the LA and RA homologous arms of E-Cad and the upstream and downstream primers. The sequences of tdTomato and eGFP were also obtained by PCR amplification. Completed the construction of the homologous recombinant plasmid pBKS-hE-Cad-tdTomato.
2. Introduced homologous recombinant plasmids into DH5-α competent cells, selected 20 single colonies and identified them.
2019.4.22-2019.4.26
1. Selected PAM sequence, constructed target plasmids.
2. Thawed and cultured WA09, A549, beas-2b, MCF7.
2019.4.26-2019.4.30
Detected the expressing levels of E-Cad & N-Cad in wild type WA09, A549, beas-2b, MCF7.
MAY
2019.5.1-2019.5.13
1. Lipofection: imported pBKS-hE-Cad-tdTomato and target plasmids into WA09, A549, BEAS-2b, MCF7.
2. Fluorescence observation, monoclonal selection, PCR amplification and identification for the target sequence.
3. Verified that the knock-in of tdTomato in A549, BEAS-2b, MCF7 failed.
4. Obtained the knock-in cell line of tdTomato in WA09.
2019.5.14-2019.5.24
The construction of LG-TetO-IN-hSnail1, pPylZ-tTR-KRAB, pPylZ-hTwist1-ERT2.
2019.5.14-2019.5.27
1. Lipofection: imported pBKS-hE-Cad-tdTomato and target plasmids again into A549, BEAS-2b, MCF7.
2. Fluorescence observation, monoclonal selection, PCR amplification and identification for the target sequence.
3. Verified that the knock-in of tdTomato in A549, BEAS-2b, MCF7 failed again.
2019.5.27-2019.5.31
Extraction of RNA from WA09-tdTomato cell line.
JUNE
2019.6.1-2019.6.7
Proved that tdTomato was inserted into the correct location in genome and cDNA levels.
2019.6.8-2019.6.21
Designed the LA and RA homologous arms of N-Cad and the upstream and downstream primers. And the sequences of eGFP were also obtained by PCR amplification. Completed the construction of the homologous recombinant plasmid pBKS-hNCad-eGFP.
2019.6.22-2019.6.28
Selected PAM sequence, constructed target plasmids.
JULY
2019.6.29-2019.7.15
Actinomycin treatment and WB analysis of protein samples: actinomycin treated WA09-tdTomato and wild-type control group.
2019.7.5-2019.7.25
Introduced homologous recombinant plasmids and target plasmids into DH5-α competent cells, selected 20 single colonies, identified .
Lipofection: imported pBKS-hNCad-eGFP and target plasmid into WA09-tdTomato.
2019.7.25-2019.7.30
1. Verified that the knock-in of eGFP in WA09-tdTomato failed
2. Chongqing meetup
AUGUST
2019.7.31-2019.8.10
1. introduced homologous recombinant plasmids and target plasmids into DH5-α competent cells, selected 20 single colonies, identified again.
2. Lipofection: imported pBKS-hNCad-eGFP and target plasmid into WA09-tdTomato again.
2019.8.10-2019.8.17
1. Completed the CAG promoter strength measurement experiment. Compared the GFP expression under the action of different promoters, including CAG, CMV, EF1a and Ubiquitin in 293 cells by flow cytometry.
2. Preparation for PPT and poster of CCIC.
2019.8.19-2019.8.24
Take part in CCIC.
SEPTEMBER
2019.8.25-2019.9.5
Verified that the knock-in of eGFP in WA09-tdtomato failed again.
Adjustment for project.
2019.9.10-2019.9.20
Treat WA09-tdTomato with TGF-beta.
2019.9.15-2019.10.2
Used lentiviral transfection to put LG-TetO-IN-hSnail1，pPylZ-tTR-KRAB and pPylZ-hTwist1-ERT2 into WA09-tdTomato separately, made a test about the relationship between efficiency and time/dosage.
2019.9.21-2019.10.3
Compared the efficiency of rtTA, TetR and tetr-krab systems. After cell transfection, treated cells with DOX for 24 hours, recorded and detected relative fluorescence expression by flow cytometry.
2019.9.20-----now
Data processing, WIKI writing, presentation preparation.
FluorescenceForFate
xiaoyang16@mails.ucas.ac.cn
Third Military Medical University,
Gaotanyan St 30, Chongqing 400038, China