Homologous recombination into the eosinophil peroxidase locus generates a strain of mice expressing Cre recombinase exclusively in eosinophils.

Homologous recombination into the eosinophil peroxidase locus generates a strain of mice expressing Cre recombinase exclusively in eosinophils.

J Leukoc Biol. 2013 Apr 29;

Authors: Doyle AD, Jacobsen EA, Ochkur SI, Willets L, Shim K, Neely J, Kloeber J, Lesuer WE, Pero RS, Lacy P, Moqbel R, Lee NA, Lee JJ

Abstract
Eosinophils are generally linked to innate host defense against helminths, as well as the pathologies associated with allergic diseases, such as asthma. Nonetheless, the activities of eosinophils remain poorly understood, which in turn, has prevented detailed definitions of their role(s) in health and disease. Homologous recombination in embryonic stem cells was used to insert a mammalianized Cre recombinase in the ORF encoding Epx. This knock-in strategy overcame previous inefficiencies associated with eosinophil-specific transgenic approaches and led to the development of a knock-in strain of mice (eoCRE), capable of mediating recombination of “floxed” reporter cassettes in >95% of peripheral blood eosinophils. We also showed that this Cre expression was limited exclusively to eosinophil-lineage committed cells with no evidence of Cre-mediated toxicity. The efficiency and specificity of Cre expression in eoCRE mice were demonstrated further in a cross with a knock-in mouse containing a “(flox-stop-flox)” DTA cassette at the ROSA26 locus, generating yet another novel, eosinophil-less strain of mice. The development of eoCRE mice represents a milestone in studies of eosinophil biology, permitting eosinophil-specific gene targeting and overexpression in the mouse as part of next-generation studies attempting to define eosinophil effector functions.

PMID: 23630390 [PubMed – as supplied by publisher]

View full post on pubmed: asthma

Comprehensive FISH Probe Design Tool Applied to Imaging Human Immunoglobulin Class Switch Recombination.

Related Articles

Comprehensive FISH Probe Design Tool Applied to Imaging Human Immunoglobulin Class Switch Recombination.

PLoS One. 2012;7(12):e51675

Authors: Nedbal J, Hobson PS, Fear DJ, Heintzmann R, Gould HJ

Abstract
We present a web engine boosted fluorescence in-situ hybridization (webFISH) algorithm using a genome-wide sequence similarity search to design target-specific single-copy and repetitive DNA FISH probes. The webFISH algorithm featuring a user-friendly interface (http://www.webfish2.org/) maximizes the coverage of the examined sequences with FISH probes by considering locally repetitive sequences absent from the remainder of the genome. The highly repetitive human immunoglobulin heavy chain sequence was analyzed using webFISH to design three sets of FISH probes. These allowed direct simultaneous detection of class switch recombination in both immunoglobulin-heavy chain alleles in single cells from a population of cultured primary B cells. It directly demonstrated asynchrony of the class switch recombination in the two alleles in structurally preserved nuclei while permitting parallel readout of protein expression by immunofluorescence staining. This novel technique offers the possibility of gaining unprecedented insight into the molecular mechanisms involved in class switch recombination.

PMID: 23272136 [PubMed – in process]

View full post on pubmed: asthma