Genetic barcoding with fluorescent proteins for multiplexed applications.
Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded 'indefinitely'. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing.
Department of Biology, San Diego State University.
Cameron A
CA
Smurthwaite
Department of Biology, San Diego State University.
Wesley
W
Williams
Department of Biology, San Diego State University.
Alexandra
A
Fetsko
Department of Biology, San Diego State University.
Darin
D
Abbadessa
Department of Biology, San Diego State University.
Zachary D
ZD
Stolp
Department of Biology, San Diego State University.
Connor W
CW
Reed
Department of Biology, San Diego State University.
Andre
A
Dharmawan
Department of Biology, San Diego State University; roland@mail.sdsu.edu.
Roland
R
Wolkowicz
D000818:Animals; D058893:DNA Barcoding, Taxonomic / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D005819:Genetic Markers / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics*; D011401:Promoter Regions, Genetic; D015202:Protein Engineering / Q000379:methods*; D012190:Retroviridae / Q000235:genetics
D005456:Fluorescent Dyes; D005819:Genetic Markers; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins