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Rboxylterminus. The amplicon was subsequently introduced into the pGEM T-Easy vector (Promega) according to the manufacturer's instructions, and the products were bidirectionally sequenced to confirm their identity. The QuikChange SiteDirected Mutagenesis Kit (Stratagene) protocol was used to perform in vitro site-directed mutagenesis to create the 95?98 kcnj2 construct. The primers used in this p
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Ementary Figure 1). All five putative transcripts contained a single open reading frame; however, only the kcnj2-12 transcript was the same length as the human KCNJ2 gene transcript (Supplementary Table 3). The structure of the KCNJ2 protein consists of two transmembrane domains (M1 and M2) linked by a pore region. The amino- and carboxyl-termini compose the cytoplasmic domain of the ion channel a
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Primers used to create the WT and mutant 95?8 kcnj2 recombinants are2. Materials and Methods2.1. Bioinformatic Analysis of Zebrafish kcnj2 Gene Orthologues. A manual reciprocal best hit approach was undertaken [22]. The human KCNJ2 amino acid sequence was queried against the zebrafish genome using the TBLASTN algorithm on Ensembl Genome Browser (Zv9 build), with search sensitivity settings on "dis
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Rboxylterminus. The amplicon was subsequently introduced into the pGEM T-Easy vector (Promega) according to the manufacturer's instructions, and the products were bidirectionally sequenced to confirm their identity. The QuikChange SiteDirected Mutagenesis Kit (Stratagene) protocol was used to perform in vitro site-directed mutagenesis to create the 95?98 kcnj2 construct. The primers used in this p
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Rboxylterminus. The amplicon was subsequently introduced into the pGEM T-Easy vector (Promega) according to the manufacturer's instructions, and the products were bidirectionally sequenced to confirm their identity. The QuikChange SiteDirected Mutagenesis Kit (Stratagene) protocol was used to perform in vitro site-directed mutagenesis to create the 95?98 kcnj2 construct. The primers used in this p
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The INFINITY Capture software system and saved as. AVI files. The number of contractions was manually counted and the number of beats per minute was calculated. The average heart rate and standard error were calculated and the results were analysed using one-way ANOVA. 2.11. Zebrafish Motility Analysis. 24 hpf dechorionated embryos were probed at the trunk of the body and yolk with fine forceps an
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D the transcribed mRNAs were purified using the lithium chloride precipitation method (according to the manufacturer's instructions). RNA quality was determined by gel electrophoresis and quantified using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc.). 100 pg of 95?8 kcnj2-12 RNA, 400 pg of WT kcnj2-12 RNA, and 100 pg each of 95?8 and WT kcnj2-12 RNA were separately injected in
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D the transcribed mRNAs were purified using the lithium chloride precipitation method (according to the manufacturer's instructions). RNA quality was determined by gel electrophoresis and quantified using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc.). 100 pg of 95?8 kcnj2-12 RNA, 400 pg of WT kcnj2-12 RNA, and 100 pg each of 95?8 and WT kcnj2-12 RNA were separately injected in