STOCK Apela^em1.1Hadj/J

Stock number: 030845
Product name: Apela^H2B-GFP 'NEO-OUT'
Research areas: Cardiovascular Research, Developmental Biology Research, Research Tools

Description

This CRISPR/Cas9 generated mutant of the Apela gene carries a H2B-GFP reporter knock-in/knock-out allele. These Apela KO reporter mice are suitable for use in applications related to the study of cardiovascular development.

Detailed description

CRISPR/cas9 endonuclease mediated genome editing of the Apela, apelin receptor early endogenous ligand, gene was used to introduce a H2B-GFP knock-in/knock-out mutation. The targeted Apela gene encodes a peptide hormone involved in embryonic cardiovascular development and embryonic stem cell self-renewal. Apela null mice exhibit low penetrance embryonic lethality, vascular development defects, extraembryonic defects. Surviving homozygous mice exhibit non-lethal cardiac defects. Approximately 10% of null embryos, by E9.5 of age, exhibit a variable phenotype of unremodeled yolk sac vasculature as well as cardiac and cranial defects. The more severely affected homozygous embryos die by E15.5. At E8.25 homozygous embryos exhibit ectopic protrusions from the anterior chorion and some thin cylindrical allantoises in the extraembryonic tissues. At E9.5 null embryos develop heart looping defects, blood pooling in the dorsal aorta, pericardial edema, and defective endothelial cell remodeling in addition to an unremodeled yolk sac vascular plexus and vitelline vessels. At E10.5 homozygous embryos have reduced somitic surface area, undeveloped somitic vasculature, and abnormal distribution of macrophages. At E15.5 surviving homozygous embryos develop a truncated right coronary vein. The reporter is detected (by GFP antibody) in the anterior endoderm and the neuroectoderm overlying the midline in E7.75 homozygous embryos . The reporter expression pattern is similar to the endogenous Apela gene expression pattern.

In an attempt to offer alleles on well-characterized or multiple genetic backgrounds, alleles are frequently moved to a genetic background different from that on which an allele was first characterized. This is the case for the strain above. It should be noted that the phenotype could vary from that originally described. We will modify the strain description if necessary as published results become available.

Development

Plasmids encoding a signal guide RNA designed to knock-in a H2B-GFP-pA reporter cassette and a loxP site flanked PGK-NEO cassette into the endogenous translational start site of the Apela gene and the cas9 nuclease were introduced into the cytoplasm of C57BL/6-Tyr^c-2J albino embryonic stem cells. Correctly targeted ES cells were injected into C57BL/6 blastocysts. The resulting male chimeric animals were crossed to CD-1 female mice for 2 generations. The mice were then crossed to transgenic mice expressing Cre recombinase under the control of the chicken beta-actin promoter coupled with the cytomegalovirus (CMV) immediate early enhancer to excise the floxed PGK-NEO cassette. The mice were bred to remove the CAG-cre transgene. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

Allele

Symbol: Apela^em1.1Hadj
Name: endonuclease-mediated mutation 1.1, Anna-Katerina Hadjantonakis
MGI accession ID: MGI:5909264
Generation method: Endonuclease-mediated
Attributes: Reporter; Null/Knockout
Synonyms: Apela^H2B-GFP; NEO-OUT
Marker symbol: Apela
Marker name: apelin receptor early endogenous ligand
Marker synonyms: Ela; Elabela; Ende; Gm10664; tdl
Chromosome: 8
Strain of origin: B6(Cg)-Tyr^c-2J
Expressed genes: GFP,Green Fluorescent Protein,
Site of expression: GFP is detected in the anterior endoderm and the neuroectoderm overlying the midline in E7.75 homozygous embryos . The reporter expression pattern is similar to the endogenous Apela gene expression pattern.
Molecular note: Plasmids encoding a signal guide RNA are designed to knock-in a H2B-GFP-pA reporter cassette and a loxP site flanked PGK-NEO cassette into exon 2, the endogenous translational start site of the gene. Cre-mediated recombination removed the floxed neo cassette. Quantitative RT-PCR confirmed that 3' UTR was not amplified although both spliced and unspliced 5'UTR is detected. This is a null allele and a reporter allele.