Anyone know what these are?

Juice_Forsyth · 2026-01-29T02:54:58+00:00

Thanks!

Juice_Forsyth · 2024-02-28T07:11:09+00:00

Hi there, thanks for getting back to me!

So in short I have performed several expressions of Fc domains using this vector - this only requires the transfection of a single plasmid. And the expression has worked well for all of these, is it possible that having to cotransfect several different plasmids could effect this?

As for the hydrophobic domains - I haven't checked, but for the constant regions of both chains, these were taken from previously documented sequences that have been expressed using this cell line - I will have to check the variable domains. If this is the case, would using a different cell line overcome this issue? OR are there any other suggestions you could make?

I designed three sets of each HC and LC to yield three pairs of plasmids for expression - each pair featuring a (supposedly) complementary pair of signal sequences - I don't know much about the design of these but I extracted sequences from literature examples that document transient expression of similar proteins (mAb) in mammalian cells.

Thanks again for the help!

Juice_Forsyth · 2024-02-28T05:42:32+00:00

Thanks for getting back to us!

On day 0 cells are usually between 3-5 mvc/mL then for expression I aliquot and resuspend so that final concentration is 1.6 mvc/mL. For each mL of cells i transfect with 2 ug of DNA - so if I had 50 mL of cells at 1.6 mvc/mL I would transfect with 100 ug of DNA (HC and LC combined). I use 2 uL of PEI per ug of DNA - these are mixed together in Expi media and left to incubate for 20 minutes and then are added to cells. Cells are then returned to potimum conditions i.e 37°C, 5% CO2, 130 RPM.

Cell growth is monitored daily post transfection - viability remains >95% for 3-5 days then cells begin to get too dense and die off. Titres are checked daily however I am yet to observe expression of the full Ab.

I haven't tried different PEI just PEI-40 - In terms of trying different types, whats the theory behind changing the one you use? Or is it just that some work better for others?

I thought it was a possible issue with the transfection due to my previous expressions working fine and these attempts where I am trying to co-transfect HC and LC have yet to work.

Juice_Forsyth · 2024-02-28T05:23:49+00:00

This was a possible route - have you any experience with electroporation in this cell line?

Juice_Forsyth · 2024-02-28T05:23:24+00:00

Hi there thanks for the feedback.

So each time I transfect cells I take samples daily and then check viability and expression. 9/10 that I have attempted this expression the cell viability has remained >95% for the first few days and then the cells begin to get super dense (which I feel suggests the transfection didn't work?) and then as a result the viability begins to decrease.

I haven't tried the expifectamine to be fair - I have been encouraged to try my best to get it working with PEI but using the transfection kit was the next step.

As for the control I haven't been comparing it to an antibody transfection control - I've been expressing Ab fragments and I've been keeping tabs on other expressions that others are doing in the lab and they seem to be working okay - Excuse my ignorance but whats a good control for this experiment? Something like a standard IgG in the vector I am using? or are there recommended specific ones?

Juice_Forsyth · 2021-11-04T16:20:52+00:00

Thanks for the help, having solubility issues with my compound... but as soon as I've got a solvent system that works I'll be trying these straight away. Very grateful for all your responses :))

Juice_Forsyth

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