Biology subjects

Fitzpatrick, D. S.

Publications and source records attributed to Fitzpatrick, D. S..

2 recordsLinked to original sources

bioRxiv2026

A Large Animal Model of Heritable Pulmonary Arterial Hypertension UsingGene-edited BMPR2 Sheep

Pulmonary Arterial Hypertension (PAH) is a rare vascular disorder characterized by elevated pressure in pulmonary arteries, eventually leading to right ventricular failure. Approximately 50% of pediatric disease and 20% of adult disease can be linked to a genetic mutation, with nearly 70% of these cases involving mutations in the bone morphogenetic protein receptor type 2 (BMPR2) locus. Investigations using rodent models have made significant advances in our understanding of BMPR2 signaling; however, limited data exist regarding the onset and course of PAH, and etiologies for phenotypic expression in these patients remain unknown. In this work, we describe the development of a novel ovine model of heritable PAH. Because homozygous disruption of BMPR2 is embryonic lethal, we developed heterozygous BMPR2 sheep by using a PAM-disrupting synonymous single stranded oligodeoxyribonucleotide alongside a single guide RNA and Cas9 mediated gene editing strategy. The resulting BMPR2(+/-) lambs demonstrated cardiac and pulmonary vascular pathology that are consistent with BMPR2 mutation-driven PAH observed in humans. Given the genetic and physiological similarities of BMPR2(+/-)sheep to humans with heritable PAH, this large animal model will serve as a vital platform for mechanistic molecular studies and will provide a much-needed pre-clinical model for extensive treatment evaluations.

molecular biology↗

bioRxiv2024

Efficient Generation of SOCS2 Knock-out Sheep by Electroporation of CRISPR-Cas9 Ribonucleoprotein Complex with Dual-sgRNAs.

Knock-out (KO) sheep were produced using CRISPR-Cas9 ribonucleoprotein complexes in zygotes targeting an 85 bp section of the first exon of the Suppressor of Cytokine Signalling-2 (SOCS2) gene. Electroporation was performed 6 hours post-fertilization with dual-guide CRISPR-Cas9 ribonucleoproteins (RNPs). Fifty-two blastocysts were transferred to 13 estrus-synchronized recipients, yielding five live lambs and one stillborn. These lambs were all compound heterozygotes with mutations predicted to result in SOCS2 KO. Three lambs carried large deletion alleles (259 bp, 1694 bp, and 2127 bp) that evaded initial detection via initial PCR screening. Off-target analysis identified a small number of mutations which may have been the result of off-target activity in regions with some homology to the guides, but notably such mutations were also observed in unedited controls. Further, we observed several orders of magnitude more mutations outside of these regions of homology in both edited animals and controls. Western blot and RT-PCR analysis of cell lines from SOCS2 KO lambs showed trace levels of SOCS2 mRNA and SOCS2 protein. In conclusion, combining IVF and electroporation of dual-guide CRISPR-Cas9 RNPs was effective at generating KO sheep.

bioengineering↗