We would like to establish tumour-bearing mice models by using a cancer cell that have been stably transfected with a plasmid. Several different groups will be established which will receive different treatments. So far, we have a control group that will not be injected with any cancer cells (normal control) and we have a control tumour group which will not receive any treatment. What we want to know, is whether we have to include control groups where the mice have been injected with the non-transfected cell line (i.e. does not contain the plasmid)? If it is needed, is it oky to just include one such group (i.e. a control tumour group not containing any plasmid and not receiving any treaments)? Or do we have to include such groups for each of the different treatments as well (i.e. a separate control tumour group without plasmid receiving treatment A, and also receiving treatment B, and also receiving treatment C and so on..)? The problem is, if this is really needed, the study gets really big, and the cost involved with that large amount of mice is too much. I hope I'm explaining this clearly. Any help or advice will be greatly appreciated!
You need to use two cell lines; first one transfected with plasmid containing your gene of interest and second with empty plasmid. Half mice are inoculated with first plasmid, another half with second. I would recommend to use vehicle treated mice as control and have at least 10 animals per treatment group. Here is groups example:
empty+vehicle (10 mice), transgene+vehicle (10 mice), empty+therapy1 (10 mice), transgene+therapy1 (10 mice), empty+therapy2 (10 mice), transgene+therapy2(10 mice) etc...
You need to use two cell lines; first one transfected with plasmid containing your gene of interest and second with empty plasmid. Half mice are inoculated with first plasmid, another half with second. I would recommend to use vehicle treated mice as control and have at least 10 animals per treatment group. Here is groups example:
empty+vehicle (10 mice), transgene+vehicle (10 mice), empty+therapy1 (10 mice), transgene+therapy1 (10 mice), empty+therapy2 (10 mice), transgene+therapy2(10 mice) etc...
if you are transfecting to create a knockdown with shRNA for example you should also include a scrambled RNA control as well as the empty plasmid. For overexpressing a gene the emply vector should be sufficient.
I agree with Dimitry Malin and Peter Thomas about the use of empty vector or scrambled Sh (i.e. mock) transfected cells as controls.
Additionally, I guess the cells were selected according to resistance to certain drugs or enriched by sorting. Please note, if a stable transfected cell line is established by clonal selection (e.g. limiting dilution, sorting etc.) then it represents only one cell (or only a group of cells) not the entire cell line population. So, you may also use two or more independently selected transfectants. This was commented by the reviewers that had evaluated one of my papers.
Thank you everyone for the comments, really appreciate it! And thank you Gűneş for the advice, I think that would be a good idea yes.
I'm sure you're quite excited, just recently experienced it myself.
We already planned our mice experiment and here's how it looks like:
- mice injected with control cell line (i.p)
- mice injected with transfected cells (overexpressing gene Y, (or knockdown cells, in that case you'd also need scrambled cells as stated above)) with groups as above..
For each group you calculate n mice => in total n*4 for control cells, plus n*4 for the transfected cells (plus n*4 of the other transfected cells if you have other cell lines to be tested; and in our case n is 4)
(Thanks to Walter Berger and Petra Heffeter who are both experienced in those experiments)
Wish you good luck.
Please, take into consideration that a DNA vector, a transfection reagent, expression of an antibiotic resistance (trans)gene, expression of a reporter (trans)gene, and selection by acute/chronic antibiotic treatment may evoke cellular responses that affect the biochemical processes under investigation. In the following review, we demonstrate that an assumption that empty vector-transfected cells preserve the cytogenetic and phenotypic characteristics, and represent the adequate control in transfection experiments is not universally valid. We exemplify a number of studies, which reported obvious genomic, transcriptomic and phenotypic changes of tumor cells after transient/stable transfection of an empty vector. Finally, we conceptualize that the diverse experimental manipulations (e.g., transgene overexpression, gene knock out/down, chemical treatments, acute changes in culture conditions, etc.) may act as a system stress, promoting intensive genome-level alterations (chromosomal instability, CIN), epigenetic and phenotypic alterations, which are beyond the function of manipulated genes.
Please see the following manuscript for details:
Stepanenko AA, Heng HH. Transient and stable vector transfection: Pitfalls, off-target effects, artifacts. Mutat Res. 2017 Jul;773:91-103. doi: 10.1016/j.mrrev.2017.05.002.
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