I want to express some variants of human anion-exchanger transporter protein in K562 cell line using viral transfection method. What is the typical workflow for achieving this. No need to go into the details of the methods. Just a typical workflow to accomplish this task is highly appreciated.
Thank you!
Depends on your labs budget. You could buy an expression vector, transfect your cells, sort or select for your marker (GFP/antibiotic), western blot and Flow-Cytometry to show presence of protein. qRT-PCR data would be good but not good enough. Then single cell sort and bam, you know have a clonal cell line established with your transporter protein.
You will probably want to begin with your protein of interest in a retro or lentiviral compatible vector. Then perform site directed mutagenesis to make the mutant of interest. You can then generate virus in HEK293T cells by transfecting the gene of interest with viral packaging plasmids. This can all be looked up on addgene.
The virus will then be collected and put onto the receiving cell line of interest.
Select for cells that received the virus with antibiotic (puromycin, hygromycin, etc.)
Western blot to confirm the process worked. It helps to have a negative control with empty viral vector transduced as well.
https://link.springer.com/protocol/10.1385%2F1-59259-394-1%3A183
http://www.jbc.org/content/270/26/15451.full
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