Generation of a conditional null allele of jumonji

Abstract

The jumonji (jmj) gene plays important roles in multiple organ development in mouse, including cardiovascular development. Since JMJ is expressed widely during mouse development, it is essential that conditional knockout approaches be employed to ablate JMJ in a tissue-specific manner to identify the cell lineage specific roles of JMJ. In this report, we describe the establishment of a jmj conditional null allele in mice by generating a loxP-flanked (floxed) jmj allele, which allows the in vivo ablation of jmj via Cre recombinase-mediated deletion. Gene targeting was used to introduce loxP sites flanking exon 3 of the jmj allele to mouse embryonic stem cells. Our results indicate that the jmj floxed allele converts to a null allele in a heart-specific manner when embryos homozygous for the floxed jmj allele and carrying the alpha-myosin heavy chain promoter-Cre transgene were analyzed by Southern and Northern blot analyses. Therefore, this mouse line harboring the conditional jmj null allele will provide a valuable tool for deciphering the tissue and cell lineage specific roles of JMJ.

Endothelial Jarid2/Jumonji Is Required for Normal Cardiac Development and Proper Notch1 Expression

“…Therefore, Jarid2 was deleted in multiple cell lineages using Cre-loxP technology to delineate the lineage-specific roles of Jarid2 in the developing heart. Our Jarid2 floxed mice (Jarid2 F/F ) that exhibited a functional loxP allele (38) were crossed with various Cre mice.…”

“…Western blotting was performed on heart tissue as described previously (38). Images were scanned and densitometry was performed using National Institutes of Health ImageJ.…”

“…For all conditional deletion of Jarid2, Jarid2 F/ϩ ; Cre/ϩ males were mated with Jarid2 F/F females. All of the mice employed for this study were bred to a mixed 129/Svj and C57BL/6 background, and genotyping was performed as described previously (3,38).…”

“…Quantitative Real Time PCR-To perform quantitative real time PCR (qRT-PCR), hearts were snap frozen in TRIzol reagent (Invitrogen), while genotyping was performed as described (3,38). Each heart was treated as an individual sample.…”

“…Antibodies used for fluorescent immunostaining were anti-NIC-1 (41) and anti-ErbB2/4 (Santa Cruz, sc-284). Jarid2 staining was performed using the Jarid2 P-100 antibody (38,42) with the TSA TM -Plus cyanine 3/cyanine 5 system (PerkinElmer Life Sciences).…”