Optimized expression vector for ion channel studies in Xenopus oocytes and mammalian cells using alfalfa mosaic virus.
Plasmid vectors used for mammalian expression or for in vitro cRNA translation can differ substantially and are rarely cross-compatible. To make comparisons between mammalian and Xenopus oocyte expression systems, it would be advantageous to use a single vector without the need for shuttle vectors or subcloning. We have designed such a vector, designated pUNIV for universal, with elements that will allow for in vitro or ex vivo expression in multiple cell types. We tested the expression of pUNIV-based cDNA cassettes using enhanced green fluorescent protein and two forms of the type A gamma-aminobutyric acid receptor (GABA(A)R) and compared pUNIV to vectors optimized for expression in either Xenopus oocytes or mammalian cells. In HEK293 cells, radioligand binding was robust, and patch clamp experiments showed that subtle macroscopic GABA(A)R kinetics were indistinguishable from our previous results. In Xenopus oocytes, agonist median effective concentration measurements matched previous work using a vector optimized for oocyte expression. Furthermore, we found that expression using pUNIV was significantly enhanced in oocytes and was remarkably long-lasting in both systems.
Venkatachalan
Srinivasan P
SP
Department of Physiology, University of Wisconsin-Madison, 601 Science Drive, Madison, WI 53711, USA.
Bushman
Jeremy D
JD
Mercado
José L
JL
Sancar
Feyza
F
Christopherson
Kelly R
KR
Boileau
Andrew J
AJ
D017794:Alfalfa mosaic virus / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D005456:Fluorescent Dyes / Q000378:metabolism; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007473:Ion Channels / Q000235:genetics*; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D007700:Kinetics; D009865:Oocytes / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010957:Plasmids; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D011869:Radioligand Assay; D011963:Receptors, GABA-A / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D013997:Time Factors; D014981:Xenopus
D005456:Fluorescent Dyes; D007473:Ion Channels; D020033:Protein Isoforms; D011963:Receptors, GABA-A; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins