Hi All.
I am using 4 different plasmid, each encoding different scFv with similar back bone, The only difference between all the plasmid is nucleotide encoding for scFv seq (Antibody Seq).
When i do transfection in HEK293T, i could see difference in transfection efficiency between all the construct, after 72hr.
Say for Example: Plasmid "A" shows 60% transfection efficiency , Plasmid B shows 45%, Plasmid "C" 30% & Plasmid D shows 10%.
Note:
1) The expression of receptor for A,B,C,D are high in HEK293T.
2) None of the scFv induce cell death -In Vitro.
3) The concentration of plasmid, the amount of transfection agent, incubation time of reaction mixture are all same for all the construct.
Hi, How do you quantify your protein? may be due to post-transnational modification.
Hello, how do you estimate the transfection efficiency: number of transfected cells or protein levels? If the latest, your 4 constructs are probably transfected on the same way but the final protein expression is different.
Best, Sandra
Hi Suresh
How different (eg in terms of GC%) is the nucleotide sequence between each sample? Is it affecting codon usage? How large is this nucleotide region that differs between each construct?
Also, is the plasmid Amp-Ori region exactly the same for each plasmid? Or do any of them contain an SV40 Ori?
Best
John
I think you should take the number of transfected cells into consideration , transfection is harsh on most cells and cell death can contribute to transfection efficiency difference, sometimes you can’t see a prominent difference under the microscope but when you quantify your protein you can see some significant changes with different transfected samples.
Suresh mentioned already no cell death. So, cells may be harvested in 48 h after transfection, and see if still the same difference in protein expression level
Thanks to all,,
Answering to Chandra
1. Hi, How do you quantify your protein? may be due to post-transnational modification.
After 72hr of transfection, I calculate transfection efficiency based on GFP expression (Reporter gene) compared with non transfected HEK293T (Negative Control) via Flow cytometry.
Ans to Sandra, % of transfected cells are calculated based on the number of cells showing GFP signal compared with control. Please find the FC image attached.
Note: GFP reporter gene for all my construct (4 different plasmid) are driven by CMV-IRES.
With this available knowledge, i assume there should be uniform expression level of GFP for 4 different plasmid. However the number of cells expressing GFP is varying between 4 different plasmid, Why it so ?
my guess is final size of the plasmid, even though the reporter is the same (so the backbone), maybe the transgene might have a difference on size and that affects at some extent the transfection efficiency.
Do different nucleotides encoding for different scFv have different bp length?
Transfections can show a lot of variability even in the same cell line and exactly the same plasmid, from time to time. One factor could be the cell density in the plates at the time of transfection. As mentioned in one of the above responses, it could be related to the size of the plasmid. It may also be useful to check the variability between experiments when you do independent repeats of the transfections using the same plasmid (eg., A) 2-3 times. One can have an internal control for transfection efficiency added to all these transfections, which is co-transfected along with the test plasmid.
Thanks to All for good discussion.
FYI -Cell density is uniform for all the construct ..
Experiment has been repeated several times , and the difference in transfection efficiency is maintained in same proportion, with plasmid B transfected cells always show higher transfection efficiency.
Plasmid A: 15E: 7963bp. (scFv -687bp) TF=32-40% GC=65%
Plasmid B: 99E: 8008bp. ( scFv -732bp) TF=53-57% GC=56%
Plasmid C: LG: 7954bp (scFv -678bp) TF=30-38% GC=67%
Plasmid D: EM: 8023bp (scFv- 747bp) TF=35-40% GC=50%
Backbone: 7276bp, which is common for all the construct.
Note:
Plasmid B always give higher transfection efficiency.
Plasmid C killed most of cells after 4-6 weeks of transfection and there where non GFP cells surviving after Antibiotic resistance.
Plasmid A Loss its GFP signal after few weeks but cells are resistance to antibiotics..
Using Internal control might be good idea to solve this riddle... Will consider.
Thanks to all ..
Hi All. I doubt my flow result, as i didn't use single stained control, live and dead cell staining control. There is a possibility to misinterpret my data.
Will repeat the exp and will let you know ..
Chitra Kannabiran , can you please share some info about the internal control that you have used when transfecting GFP expressing plasmid please ..
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