I would like to overexpress a gene in cell culture model. I have never done this before and from what i have read so far it typically involves establishing a stably expressing cell line usually via retroviral methods. I currently dont have the experience to do such an experiment and so was wondering are there any commerical options available to me whereby i could give the sequence of the gene to the company and they generate the cell line for me. Or if there are other suggestions about what may be the easiest straightforward method for me to achieve my goal would be appreciated.
Well, you dont have to estabilish a stably expressing cell line, if you want to see the behavior of your over expressed protein inside the cell line. For transient expression of your protein (human?) you can simply clone it in pcDNA vector(driven by CMV promoter), and transfect them in a mammalian cell line (eg:HEK 293T). 24 or 48 h post-transfection, lyse the cells and do your assays with the protein in the cell lysate. It all depends on what you want to do further. Expression of two or more proteins using the respective plasmids can also be done using this.
Well, you dont have to estabilish a stably expressing cell line, if you want to see the behavior of your over expressed protein inside the cell line. For transient expression of your protein (human?) you can simply clone it in pcDNA vector(driven by CMV promoter), and transfect them in a mammalian cell line (eg:HEK 293T). 24 or 48 h post-transfection, lyse the cells and do your assays with the protein in the cell lysate. It all depends on what you want to do further. Expression of two or more proteins using the respective plasmids can also be done using this.
HEK cells are little divas and compared to the output they are expensive. I would go for insect cells like SF21 or Sf9 and use the baculovirus system. However it depends on your protein. Maybe an E. coli based system is sufficient ...
Thanks for the answers they have given me some things to think about. I want to express the gene in a mouse muscle cell line and assess what it does to the phenotype of the cells. If i want to put the sequence of the gene into a vector how do i make sure it is in the correct read frame or what factors need to be considered in relation to to this issue.
If you don't need stable expression, use a suitable expression vector for eukariotic cells, preferrable with a resistence which you can select for. 293T cells are not so good for this, since they have already been stably transfected and carry a G418 resistence. I found them not to hard to transfect, but they have the disadvantage of the resistence. Native 293 cells on the other hand grew usually quite slow.
You take a suitable program like Serial Cloner (free, multiple OS), GENtle (free, unstable), other comercially available tools or even web applications. Then you import the sequence of your target vector and the sequence of your gene or source plasmid. Now you can perform in silico PCR-, restriction- and ligation-reactions. Afterwards you check the AS-sequence of your freshly ligated plasmid. Are you going to use a tag? Then it would be important to check for misplaced stop codons and for the starting methionine of gene and tag. Using the programs you can manually change the reading frame of your DNA-sequence. If both "Met" are present in the same reading-frame, then your plasmid should do its job.
What other factors? Concerning the cloning or the expression?
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