Our laboratory has two areas of interest – prostanoid biology and the role of peripheral molecular clocks in cardiovascular biology, metabolism and aging. Perhaps the distinguishing feature of our groups is that we pursue interdisciplinary translational science with a focus on therapeutics. Thus, we work in different model systems – mammalian cells, worms, fish and mice – but also in humans. Ideally we develop quantitative approaches that can be projected from our experiments in the model systems to guide elucidation of drug action in humans. To this end, we have long utilized mass spectrometry, initially to target the arachidonate derived lipidome, but more latterly also the proteome.
Currently, we are interested in several aspects of prostanoid research. We utilize a remarkably broad array of mutant mice to elucidate the biology of the two COX enzymes and the prostanoid receptors. We are particularly interested in the comparative efficacy and safety of pharmacological inhibition of COXs versus the microsomal PGE synthase– 1. We are interested in the potentially countervailing actions of prostanoids on stem cell differentiation and in elucidating the broader cardiovascular biology of prostaglandins D2 and F2α. Finally, besides inhibitors of mPGES–1 we are interested in the translational therapeutics of various receptor antagonists, aspirin and fish oils.
In the area of clock biology, we are probing the role of the clock in aging in mice and worms and using cell specific deletions of core clock components to look at how communication paradigms between discrete peripheral clocks influence cardiovascular biology and metabolism. Finally, we are taking systems approaches to investigate how perturbation of peripheral clocks result in central clock dependent phenotypes.
Finally, we are involved in the interdisciplinary PENTACON consortium designed to integrate basic and clinical research in 5 systems – yeast, mammalian cells, fish, mice and humans ( both in detail and at scale) – with the objective of predicting NSAID efficacy and cardiovascular hazard in patients.
Teegarden, Sarah., PhD
Role: Associate Director, FitzGerald Lab
Bmal1 KO in adipocytes
To generate adipocyte-specific deletion of Arntl, mice was bred with a conditional Arntl ((also known as Bmal1 or Mop3) allele with mice expressing Cre recombinase under the control of the adipocyte-specific promoter of the adipocyte protein 2 (aP2) gene (aP2-Cre mice)
Brown Adipocyte-specific deletion of Bmal1(BA-Bmal1-KO) mice were generated by crossbreeding Bmal1flox/flox (Jackson Laboratory, stock# 007668) with UCP-1 driven Cre mice (in the C57BL/6 background)
Global Bmal1 KO
COX genes switched
A COX-1>COX-2 mouse strain on a mixed C57BL/6×129/Sv genetic background was established previously by replacing most of exons 1–9 of Ptgs2 with the entire Ptgs1 cDNA and cryo-recovered from JAX (#008104). These mice were backcrossed with the C57BL/6 strain for 10 generations. Cross-breeding of COX-1>COX-2 and COX-2>COX-1 mice produces Reversa mice.
A neomycin resistance cassette was inserted into the middle of intron 10 of the PGHS1 (COX-1) gene by targeting in embryonic stem cells. Insertion of this cassette within intronic sequences has previously been shown to generate a hypomorphic allele or knockdown of gene expression to very low levels.
COX-1 > COX-2 mice were generated by replacing most of the coding region of Ptgs2 (exons 1-9) with Ptgs1 cDNA while leaving the Ptgs2 promoter region and 3’-UTR intact through conventional gene targeting technology.
We introduced a point mutation (1121a→t) into the Ptgs2 gene (which encodes PGHS2) of mice by gene targeting, resulting in a Y385F substitution, to create a genetic mimic of selective COX-2 inhibition (cycloxygenase activity is inhibited, but peroxidase activity is intact). We numbered the tyrosine residue of PGHS2 in accordance with the numbering of sheep PGHS1, the recognized standard for numbering amino acid residues of PHGS isoforms. Accordingly, the actual position of the affected tyrosine residue in mouse PGHS2 is Tyr371 based on numbering the amino-terminal methionine of the signal peptide as residue 1.
COX2 knocked into COX1 locus
COX-2>COX-1mouse was created by partial replacement of the coding region of (COX1) Ptgs1 (exons 3–10) with the targeting vector, while leaving the Ptgs1 promoter region (exons 1 and 2) and the 3′-untranslated region (UTR) intact.
The DP1 KOs were a kind gift from Dr. Shuh Narumiya of the University of Kyoto. DP1 KO (C57BL/6J and Sv129) mice were intercrossed with fully backcrossed LDLR KOs.
EC COX2 KO
Mice with the COX-2 gene flanked with two LoxP sites, COX-2F/F mice, were crossed with C57BL/6 Tie2-Cre mice to obtain EC COX-2−/− mice.
global FP (Ptgfr) KO
Gift from S. Narumiya (Kyoto University, Sakyo-ku, Kyoto, Japan)
Global DP1 KO
The DP1 KOs were a kind gift from Dr. Shuh Narumiya of the University of Kyoto.
Tamoxifen inducible global Bmal1 KO.
The Bmal1f/f floxed mice were crossed with a tamoxifen-inducible universal Cre (EsrCre) mice or NestinCre mice (Jackson Laboratories, Bar Harbor, ME) to yield Bmal1f/f-EsrCre mice and Bmal1f/f-NestinCre (nKO) mice, respectively. To generate inducible Bmal1 knockout (iKO) mice, 3m old (unless specified) Bmal1f/f-EsrCre mice were treated with tamoxifen as previously described.
Exons 6, 7, 8 and of COX-2 (PTGS2) were flanked by two directly repeated loxp sites inserted into the corresponding introns. These mice were then crossed into merCremer mice under the alpha-myosin heavy chain promoter to permit tamoxifen-dependent deletion of COX-2 in cardiomyocytes.
Mice with the COX-2 gene flanked by two LoxP sites were crossed with mice expressing LysM Cre (Cre recombinase in myeloid cells). These mice were then crossed with LDLR knockout mice (The Jackson Laboratory, Bar Harbor, ME).
Mice with the COX-2 gene flanked with two LoxP sites were crossed with LysM-Cre mice to delete COX-2 in myeloid cells, including macrophages, neutrophils, and some dendritic cells.
Mcpt5Cre/COX2 KO/LDLR KO
mPGES-1 KO mice (Trebino et al. 2003) were crossed with LDLR KO mice (Jackson Laboratory).
Cardiomyocyte-specific COX2 KO
Floxed COX-2 mice were generated on a C57/BL6 and 129SV mixed genetic background by inserting loxP sites in introns 5 and 8 of the COX-2 gene via DNA homologous recombination. The α- myosin heavy chain promoter was used to restrict merCremer expression to Cardiomyocytes.
The IPKO mice were generated by homologous recombination in embryonic stem (ES) cells.
Mice with the COX-2 gene flanked with 2 LoxP sites, COX-2f/f mice on a mixed 129Sv/B6 background (∼75% B6, ∼25% 129Sv) were crossed with B6 CD4Cre mice (Taconic, Hudson, NY) to obtain CD4Cre/COX-2f/+ mice, which then were backcrossed with COX-2f/f to create CD4Cre/COX-2f/f mice. These mice were crossed with COX-2f/f to generate CD4Cre/COX-2f/f mice as T cell-specific COX-2-deficient mice (TCKOs).
Global TP receptor KO
Tp–/– were kindly provided by Prof Thomas M. Coffman from the Department of Medicine at the University of North Carolina
Mice with the COX-2 gene flanked with two LoxP sites were crossed with mice expressing SM22-Cre (vascular smooth muscle cells) or Tie2-Cre (endothelial cells). Double knockout mice lacking COX-2 in both vascular cell types were produced by crossing these two lines together.