I want to be able to control the expression of protein of interest in 4T1 cell line and wanted to check if there could be a threshold of expression that determines the efficacy of vaccination based approach.
Could anyone help me with any literature and procedures pertaining to such modification as I am a complete beginner.
What I plan to do is:
1. Isolate the cDNA and clone it in some kind of expression vector. Can such expression vector have inducible promoters?
2. Use these vectors to transfect the 4T1 cells (preferably retrovirus mediated transfer).
3. And use the transfected 4T1 cells to assay for efficacy of vacciination based approach.
I will appreciate any information regarding such process.
I stopped cloning 2 decades ago and I never worked with 4T1 cells - but from reading the original abstract, they appear to be an attractive model for elucidating different steps of tumor metastasis. I would expect the existence of all kinds of expression vectors including inducible ones - I would suggest checking some of the webpages of companies, or wait a few days for further answers here.
I can recommend retrovirus-mediated gene transfer with Moloney Murine Leukemia virus vectors, as described in my attached paper (it should include some of the original references, e.g. from C. Cepko). Those vectors included a selection marker for G418 resistance.
Article A model for primitive neuroectodermal tumors in transgenic n...
Invitrogen has the TET-OFF, TET-ON system for inducible control of genes (by tetracycline). It works wonderfully, It is however not trivial to make your final cells, because you need few transfection/transduction steps (download the manual, it is useful) plus cloning. For cloning your cDNA, I would use the expression plasmid Invitrogen recommend (pCNDA6, I think) because then your construct is immediately suitable for the next steps required for the inducible system production (Tet inducible), but also for standard transient transfection, in case.
The most laborious step in the procedure is the introduction of the FRT site into your cells. The site is needed for recombining your gene of interest into the genome of your cell. You could check in the Invitrogen-site or if some researcher have already the 4T1 cell with the frt . II quickly searched in PubMed and in this publication (http://www.biomedcentral.com/1471-2407/8/228) the authors seem to have one such modified line.
good luck
PS consider subcontracting your cloning to a company
PPS if you get cells from another lab, AUTHENTICATE THEM
4T1 cells don't transfect particularly well, but they seem to transduce quite well with lentivirus. One thing we have found is that virus-mediated expression was more efficient for certain cell morphologies (since 4T1s seem to exist as a mixture of morphologies). We would infect the cells and see decent transduction (50-60%?) I then treated the infected cells for 48h with sodium butyrate and saw blazing GFP, 100% positive transduction. It was like some of the cells could be infected with LV, they just didn't express any protein until I added the sodium butyrate. They're a funny cell line, so maybe keep that in mind.
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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