PMC:4608092 / 14502-17496
Annnotations
{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/4608092","sourcedb":"PMC","sourceid":"4608092","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4608092","text":"Vascular EC- or SMC-Specific Rnf213 Tg Mouse Production\nThe transgene construct consisted of the following components: cysteine-adenine-guanine (CAG) promoter–LoxP–PGK promoter–Neo–3 SV40 poly(A) sequences–LoxP–mouse Rnf213 (WT or R4757K mutant) coding sequence–beta globin polyA (Figure2). The construct was generated using Gateway technology (Invitrogen). An entry vector harboring mouse Rnf213 WT was produced based on the pENTR4 vector. Five fragments of the Rnf213 coding sequence were amplified by RT-PCR from C57BL/6 mouse liver RNA using the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (Invitrogen) using primers, which are shown in Table S2. Fragments 1 and 2 were connected using the XhoI site at 3259 to 3264 bp. Fragments 2 and 3 were connected using the EcoRI site at 6080 to 6085 bp. Fragments 3 and 4 were connected using the SacII site at 8975 to 8980 bp. Fragments 4 and 5 were connected using the MluI site at 12 651 to 12 656 bp. The full-length Rnf213 coding sequence was integrated into the pENTR4 vector using NcoI and NotI sites. An allelic ortholog of human p.R4810K (p.R4757K) was introduced into Rnf213 WT using a site-directed mutagenesis kit (Invitrogen). A donation vector was generated based on the PGKneotpAlox2 vector, containing the LoxP–PGK promoter–Neo-3 SV40 poly(A)–LoxP cassette. The CAG promoter and beta-globin polyA were amplified using the pCAGGS vector as a template. The Gateway recombination cassette (attR1-ccdB-attR2) was amplified using the pDEST vector as a template. The CAG promoter was cloned into the PGKneotpAlox2 vector, upstream of the first LoxP using SacI and NotI sites. The AttR1-ccdB-attR2 cassette and beta-globin polyA were introduced into the PGKneotpAlox2 vector, downstream of the second LoxP using NheI and XhoI sites and XhoI and KpnI sites, respectively. The entry and donation vectors were converted by an LR plus clonase reaction to produce the transgene construct.\nFigure 2 Schematic diagram of Rnf213 conditional expression in ECs or SMCs. CAG indicates cysteine-adenine-guanine; ECs, endothelial cells; SMCs, smooth muscle cells; WT, wild type. The transgene constructs were digested with PvuI and KpnI, and a DNA fragment of 20 kb was purified and then microinjected into fertilized C57BL/6 mouse eggs to generate Tg mice. Genotypes of Tg offspring were determined by PCR using the primers shown in Table S3. To obtain mice harboring vascular ECs or SMCs overexpressing Rnf213, Tg founders were bred with mice expressing a Cre transgene driven by either the Tie2 kinase promoter/enhancer (Tek; strain name: B6.Cg-Tg(Tek-cre)12Flv/J) or the smooth muscle protein 22-alpha promoter (strain name: B6.Cg-Tg(Tagln-cre)1Her/J; The Jackson Laboratory, Bar Harbor, ME). Specific expression in ECs or SMCs was confirmed by western blotting. ECs were purified from lungs by magnetic cell sorting using anti-CD31/PECAM-1 antibody (Life Technology, Grand Island, NY). Aorta was used as an SMC source.","divisions":[{"label":"Title","span":{"begin":0,"end":55}},{"label":"Figure caption","span":{"begin":1965,"end":2149}}],"tracks":[]}