Localized transfection on arrays of magnetic beads coated with PCR products.
High-throughput gene analysis would benefit from new approaches for delivering DNA or RNA into cells. Here we describe a simple system that allows any molecular biology laboratory to carry out multiple, parallel cell transfections on microscope coverslip arrays. By using magnetically defined positions and PCR product-coated paramagnetic beads, we achieved transfection in a variety of cell lines. Beads may be added to the cells at any time, allowing both spatial and temporal control of transfection. Because the beads may be coated with more than one gene construct, the method can be used to achieve cotransfection within single cells. Furthermore, PCR-generated mutants may be conveniently screened, bypassing cloning and plasmid purification steps. We illustrated the applicability of the method by screening combinatorial peptide libraries, fused to GFP, to identify previously unknown cellular localization motifs. In this way, we identified several localizing peptides, including structured localization signals based around the scaffold of a single C2H2 zinc finger.
Isalan
Mark
M
EMBL, Meyerhofstrasse 1, Heidelberg D-69117, Germany. isalan@embl.de
Santori
Maria Isabel
MI
Gonzalez
Cayetano
C
Serrano
Luis
L
D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D020099:Coated Materials, Biocompatible / Q000737:chemistry*; D020869:Gene Expression Profiling / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D008280:Magnetics; D008422:Materials Testing; D051379:Mice; D008863:Microspheres; D041681:NIH 3T3 Cells; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D019151:Peptide Library; D016133:Polymerase Chain Reaction / Q000379:methods*; D014162:Transfection / Q000379:methods*
D020099:Coated Materials, Biocompatible; D019151:Peptide Library