In order to verify the effectiveness of our system and components, we designed and carried out multiple experiments including sequencing, plasmid transfection, Western blot, RT-PCR, etc., so as to verify whether the plasmid sequence, the expression of target gene transcript after plasmid transfection, and the expression of target gene expression products were consistent with the design and expectation.
Plasmid Vector Validation
We have ordered several plasmids from GENECHEM. The plasmids are numbered as: CV236-pAlbu-CopGFP, CV236-pAlb-tTS-rtTA-EGFP, CV236-TRE-miniCMV-mCherry, GV219-KIBRA-EGFP, GV219-nSMase2-EYFP, GV219-SDC4-2A-STEAP3-2A-NadB-EYFP, Pcmv-iRGD-MIR155-PD-L1-CD47-KRAS, PcDNA3.1(-)-PCMV-IRGD-mir155-CD47, pcDNA3.1(-)-PCMV-IRGD-mir155-KRAS, pcDNA3.1(-)-PCMV-IRGD-mir155-PD-L1, plasmids were transported in the form of genetically engineered Escherichia coli strains preserved in glycerol. The DNA sequence of the plasmid was verified by the steps of strain resuscitation, bacterial culture, plasmid extraction, and sequencing.
Validation of Vector Effectiveness
1. Plasmid transfection
We transfected the above plasmids into HepG2 and 293T cell lines for verification, respectively. The purpose of transfection was to verify whether the target gene could be expressed normally.
2. Rt-PCR & qPCR
Total RNA was extracted 24 hours after transfection for reverse transcription, followed by fluorescence real-time quantitative PCR (qPCR) to detect the transcription expression of the target gene. To subtract background expression and compare between different groups, GAPDH was selected as the reference gene, and the MOCK group (blank control) and the NC group (negative control) were added.
3. Western Blot
Total protein was extracted 36 h after transfection for Western Blot assay to quantify the final product of target gene expression.
4. Exosome detection
We designed components to promote exosome release and, therefore, we collected exosomes from 293T cell cultures transfected with associated plasmids. Cell culture medium was extracted 36 h after transfection for exosome extraction. After calculation, it was found that the concentration of exosomes increased, and the particle size of exosomes did not change much.