Transfection Round 2
Bhuvi M -
Hello everyone! Last week, I discovered that we had mixed up some of the antibiotics we used for our transfected cells.
We conducted two transfections: one involving Axl into OE33 and the other MAL into Flo-1. I was personally overseeing the Axl transfection for my project, while the MAL transfection was conducted for a separate project.
Despite using the correct antibiotic for OE33, our cells still experienced mortality. This indicates either an unsuccessful transfection or the induction of apoptosis by the added gene. If apoptosis was triggered, it would imply that Axl isn’t responsible for afatinib resistance in esophageal adenocarcinoma. To validate this, we are reattempting the transfection with fresh cells.
I plated the cells yesterday, with 3*10^5 cells in each well, and we will monitor until they reach 80-90 percent confluency.
Unfortunately, we used the wrong antibiotic for selecting Flo-1 cells with MAL. Consequently, I am redoing the transfection. Flo-1, a more aggressive and invasive esophageal adenocarcinoma cell line, was briefly discussed in my blog post on the invasion assay. MAL is a gene known to regulate membrane order and transport vesicle docking, thus influencing cell signaling.
Another experiment I’m exploring involves inhibiting Axl in GD, an afatinib-resistant cell line, requiring a knockdown assay. In this assay, we distribute an equal number of cells across six wells and administer varying amounts of the inhibition drug. Determining the optimal drug dosage is crucial, as too much would induce cell death while too little would yield no effect.
Inhibition essentially silences the gene in the resistant cell line, contrasting the gene addition process of transfection in parental cells.
Additionally, this week, I’ve been compiling data from the cBioPortal to identify activated genes in patients developing esophageal adenocarcinoma.
Thank you for reading!