Optimized expression vector for ion channel studies in Xenopus oocytes and mammalian cells using alfalfa mosaic virus

Abstract

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 γ-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.

Structural Mechanisms Underlying Benzodiazepine Modulation of the GABAAReceptor

“…Rat cDNA encoding ␣ 1 , ␤ 2 , and ␥ 2L receptor subunits in the pUNIV vector (Venkatachalan et al, 2007) were used for all molecular cloning and functional studies. This vector [kindly provided by S. Venkatachalan and A. Boileau (Department of Physiology, University of Wisconsin-Madison, Madison, WI)] is advantageous, because it can be used for expression in Xenopus oocytes and mammalian human embryonic kidney 293 (HEK293) cells without further subcloning.…”

“…HEK293T cells were grown in minimum essential medium with Earle's salts (Invitrogen, Grand Island, NY) containing 10% fetal bovine serum in a 37°C incubator under 5% CO 2 atmosphere. For binding experiments, cells were plated on 100 mm dishes and transiently transfected with pUNIV-␣ 1 , pUNIV-␤ 2 , and either pUNIV-␥ 2 or mutant pUNIV-␥ 2 DNA at ϳ40% confluency (Venkatachalan et al, 2007) using a standard CaHPO 4 precipitation method (Graham and van der Eb, 1973). In general, cells were transfected with equal ratios of subunit DNA (4 g/subunit).…”

Different Residues in the GABAAReceptor Benzodiazepine Binding Pocket Mediate Benzodiazepine Efficacy and Binding

“…Rat cDNAs encoding the GABA A R ␣ 1 , ␤ 2 , and ␥ 2L subunits in the pUNIV vector (Venkatachalan et al, 2007) were used. Cysteine mutations in the ␣ 1 and ␥ 2L subunits were made previously using recombinant polymerase chain reaction and verified by doublestranded DNA sequencing.…”

In Vitro and In Vivo Evidence for Active Brain Uptake of the GHB Analog HOCPCA by the Monocarboxylate Transporter Subtype 1

“…Constructs encoding rat MCT1, -2, and -4 or rat embigin in the pGHJ vector were kindly provided by Professor A. Halestrap, University of Bristol, UK. All initial uptake studies were conducted with these constructs, but for mutational studies, the cDNAs were subcloned by polymerase chain reaction into the pUNIV vector (Addgene, Cambridge, MA), reported to ease cRNA transcription and give higher expression levels in Xenopus oocytes (Venkatachalan et al, 2007). All forward primers contained the alfalfa mosaic virus sequence segment (in italic), the MCT coding sequence (in bold), as well as the NheI restriction site.…”