Journal: Basic Research in Cardiology
Article Title: Regulator of G protein signalling 14 attenuates cardiac remodelling through the MEK–ERK1/2 signalling pathway
Figure Lengend Snippet: Schematic diagram of the construction of RGS14 -KO mice using the CRISPR-Cas9 method and identification of RGS14 expression. a One sgRNA targeting a region downstream of the 3′ end of exon 3 in the RGS14 mouse gene was designed and constructed. b After microinjection, a T7E1 assay indicated that four out of six pups contained cleavage products, suggesting a mixture of mutant and wild-type DNA templates in these mice. c Following subcloning of the PCR products, eight subclones of each mouse were sequenced. All of the subclones carried a single mutant allele, whereas two indels (#5–5, #5–6) produced frameshift mutations. Founder #5–5 was mated to a C57BL/6J mouse to obtain the F1 generation. d Gene sequencing for RGS14 expression levels in hearts from wild-type and RGS14 knockout groups. e Representative western blots for RGS14 expression levels in hearts from the RGS14 +/+ and RGS14 −/− groups
Article Snippet: Cardiac-specific RGS14 -overexpressing mice Full-length mouse RGS14 Complementary DNA (cDNA) (OriGene, MC204443) was ligated into the chicken β-actin gene (CAG) promoter expression vector, which was linearized and purified using the QIAquick Gel Extraction Kit (Qiagen, 28704).
Techniques: Mouse Assay, CRISPR, Expressing, Construct, Mutagenesis, Subcloning, Polymerase Chain Reaction, Produced, Sequencing, Knock-Out, Western Blot