Anything that is fairly cheap, good expression levels and can produce stable transfections of cell lines such as HCT116 and COLO205.
Why don't you use Addgene.
http://www.addgene.org/ordering/
pCS vectors are very strong and has a bleo selection marker.
http://www.addgene.org/12158/
You can generate the retrovirus with this plasmid, but also transfect this plasmid directly like other expression vectors. It may be too strong depending on your experiments and/or proteins you want to express. But you can search other plasmids from addgene. Some have GFP inserted in the multi-cloning site, but it can be good a negative control and you can replace it with gene you are interested in.
I suggest Nano-Vectors such as Gold Nano Rod or Nano-Peptides (cell penetrating peptides). The transaction and release systems in these such vectors are much more controllable, detectable and precise.
if all u need is stable cell line, pCDNA3 series are all good. after you have your construct inserted into the plasmid, cut it to linear ( keep G418 marker and your insert intact), transfect the linear DNA to your cells (with whichever method you use) and G418 select for colonies. it takes about 2-3 weeks for G418 selection to get any descent size colonies.
I agree with Kazuo Fushimi. You can start searching addgene. It's a place where people share plamids. You only need to pay $60 each plasmid for and handling. Basically you can find lentiviral, retroviral plasmid backbones for your cells. If your gene is not that new, you might be able to find the plasmid with your gene's ORF in it. The only problem is you have to be in a non-profit institute to get the price.
Hi Anne-Marie,
We used two vectors for stable expression in colorectal cell lines. I suggest you pBabepuro which is a retroviral vector that you can buy at Addgene (http://www.addgene.org/1764/). You will need retroviral packing vector though. Otherwise, we use a modified version of the Invitrogen pLenti5 vector, which is a lentiviral expression system. If you need, I could see if we can send it to you.
Good luck.
Jean-Philippe
I have never transfected HCT116 and COLO205, but I have transfect several other colon cell lines. The transfection efficiency varies strongly from cell line to cell line. Also, you need to optimize the transfection (i.e. use several different transfection reagents). As far as vectors are concerned I've used SV40 promoter containing vectors such as pCDNA3 for overexpression constructs. The family of knockdown shRNA vectors in pLKO1 work well in our hands. However, in the long run, how stable the knockdown is, is still pretty much a mystery and veries (perhaps) from gene to gene.
I've transfected miRzip based vectors (SBI) into HCT116, DLD1, and SW480 by lipofectamine2000. After couple of months culturing them with puromycin (selection pressure), I got my target gene stably expressed in these colon cancer cell lines even when I withdrawed puromycin.
Thanks a lot for your answers everyone, I'll take a look at your suggestions :)
pLVX-IRES-puro vector is a lentiviral vector from Clontech that I've already used to stably transfect CRC cell lines such as NCI-H508, DiFi and HDC-142 cells with very good results...
I can't speak directly to colorectal cancer cell lines, but we have had tremendous success with phCMV1 from Genlantis. It has a G418 selection marker and we have used it to transiently and stably transfect several different human cell lines. It is smaller than pcDNA3 series and appears to express cDNA's at much higher levels. I used it to overexpress full length BRCA2 in 293Ts for purification and it worked great. see Nature. 2010 Oct 7;467(7316):678-83.
Purified human BRCA2 stimulates RAD51-mediated recombination.
Jensen RB, Carreira A, Kowalczykowski SC.
Try lentiviral vectors. Also contact Paul Clarke at the ICR in London for details of construct particulars.
We have had good success with pcDNA-3.1 G418 selection. The invitrogen Topo vectors are extremely easy for insertion of your gene/peptide of interest. Not sure of the cells you are using, but Tech Support at Invitrogen would help.
Thanks Divya and Ralph, I may be able to borrow some pcDNA3 vectors from work colleagues.....
We are colon cancer lab and have transfected HCT116 as well as ~20-30 other CRC lines (but not COLO205) with nice success in terms of stable expression using the pcDNA3.1 series of vectors. We have found that the effectiveness of the transfection reagent will vary with cell line, but most of the time Fugene from Roche or Effectene from Qiagen work well for CRC lines. For HCT116, both work nicely.
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