ASIC1a Mutant Expression in Xenopus

The human ASIC1a cDNA construct (García-Añoveros et al., 1997 (link)) was cloned into a pSP65 vector containing 5’- and 3’- untranslated sequences for expression in Xenopous oocytes. As reported recently, this construct contains the mutation G212D (Vaithia et al., 2019 (link)). Point mutations were introduced by site-directed mutagenesis using KAPA HiFi HotStart PCR polymerase (Roche Diagnostics, Rotkreuz, Switzerland). All mutations were verified by sequencing (Synergen Biotech). Complementary RNAs were synthetized in vitro using the mMESSAGE mMACHINE SP6 kit (Thermofisher).

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Vullo S., Ambrosio N., Kucera J.P., Bignucolo O, & Kellenberger S. (2021). Kinetic analysis of ASIC1a delineates conformational signaling from proton-sensing domains to the channel gate. eLife, 10, e66488.

Publication 2021

KAPA HiFi HotStart PCR polymerase mMESSAGE mMACHINE SP6 kit

Cdna Complementary rnas Diagnostics Human Mutations Oocytes Point mutations Site directed mutagenesis Vector

Corresponding Organization : University of Lausanne

Other organizations : University of Bern, SIB Swiss Institute of Bioinformatics

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Variable analysis

independent variables

Point mutations introduced by site-directed mutagenesis using KAPA HiFi HotStart PCR polymerase

dependent variables

Not explicitly mentioned

control variables

Xenopous oocytes used for expression of the human ASIC1a cDNA construct
5'- and 3'- untranslated sequences of the pSP65 vector
Complementary RNAs synthesized in vitro using the mMESSAGE mMACHINE SP6 kit

controls

Positive control: Not specified
Negative control: Not specified

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