WZsGreen/+: a new green fluorescent protein knock-in mouse model for the study of KIT-expressing cells in gut and cerebellum.
In the small intestine, interstitial cells of Cajal (ICC) surrounding the myenteric plexus generate the pacemaking slow waves that are essential for an efficient intestinal transit. The underlying molecular mechanisms of the slow wave are poorly known. KIT is currently the sole practical marker for ICC. Attempts to purify living ICC have so far largely failed, due to the loss of the KIT epitope during enzymatic dissociation. Aiming to identify and isolate living ICC, we designed a knock-in strategy to express a fluorescent tag in KIT-expressing cells by inserting the sequence of the novel green fluorescent protein ZsGreen into the first exon of the c-Kit gene, creating a null allele called W(ZsGreen). In the gastrointestinal tract of heterozygous W(ZsGreen)/+ mice, tiny ZsGreen fluorescent dots were observed in all KIT-expressing ICC populations, with exception of ICC at the deep muscular plexus in small intestine. During development of the gastrointestinal tract, ZsGreen expression followed KIT expression in a spatiotemporal way. Stellate and basket KIT-expressing cells in the molecular layer of the cerebellum also exhibited ZsGreen dots, whereas no ZsGreen was detected in skin, testis, and bone marrow. ZsGreen dot-containing intestinal cells could be isolated from jejunum and maintained alive in culture for at least 3 days. ZsGreen is a suitable alternative to EGFP in transgenic animals. The novel W(ZsGreen)/+ model reported here appears to be a promising tool for live studies of KIT-expressing cells in the gastrointestinal tract and cerebellum and for the further analysis of pacemaker mechanisms.
Wouters
Mira
M
Johnson and Johnson, Pharmaceutical Research and Development, A Subdivision of Janssen Pharmaceutica, Department of Gastrointestinal Pharmacology, Beerse, Belgium.
Smans
Karine
K
Vanderwinden
Jean-Marie
JM
D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002531:Cerebellum / Q000378:metabolism*; D002522:Chlorocebus aethiops; D020541:Coiled Bodies / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D005786:Gene Expression Regulation; D005838:Genotype; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D007583:Jejunum / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D019009:Proto-Oncogene Proteins c-kit / Q000096:biosynthesis*; D011993:Recombinant Fusion Proteins / Q000235:genetics*
D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; C510869:W(ZsGreen) protein; D049452:Green Fluorescent Proteins; D019009:Proto-Oncogene Proteins c-kit