Generation and Characterization of a Novel CD19-CherryLuciferase (CD19CL) Mouse Model: A New Fluorescent/Bioluminescent Model for the Study of B-Cell Development and Lymphomagenesis

Abstract 3940

Existing mouse models available to study the effects of new treatment strategies for lymphoma almost exclusively rely on xenograft models of the disease. These models, while informative, do not mirror the natural history of a disease that arises in the bone marrow or lymphatic system, as seen in patients. While spontaneous models may be less contrived, they pose numerous obstacles regarding the optimal strategies to image changes in tumor volume as a function of time and or treatment. The prospect of integrating both fluorescence and bioluminescence detection capabilities into a single genetically engineered mouse (GEM) model may offer a unique opportunity to assess this biology in a more realistic, cost-effective, and efficient manner. We first constructed a fusion protein consisting of the monomeric mutant red fluorescent mCherry, and the synthetic-firefly Luciferase by cloning the mCherry gene into the plasmid vector pGL4.13[luc2/SV40] (Promega) carrying the luciferase gene, thus obtaining the pGLCherryLuciferase plasmid, where the Cherry and the luciferase genes formed one open reading frame. Analysis of pGLCherryluciferase transfected human embryonic kidney 293 (HEK 293) cells via flow cytometry and luciferase activity confirmed that the cherryluciferase fusion protein retained its dual bioluminescent/fluorescent activity in vitro. In order to express the Cherry-Luciferase fusion protein in cells of the B lineage in mice we then generated a CD19cherryluciferase targeting vector, a modified version of the CD19cre targeting vector (Rickert R C et al, Nature 1995), where a cre/neo gene cassette was substituted by a Cherry-luciferase/neo cassette and the 330 bp fragment upstream of exon1 by a 1741 bp fragment obtained by PCR-amplification of ES(E14-1)-cell DNA using oligonucleotides tagged with restriction sites at their 5' end. Positive clones of transfected mouse embryonic stem (ES) cells were injected into C57BL/6J blastocysts to obtain the CD19cherryluciferase transgenic mouse, which retain one functional CD19 allele. Characterization of the transgenic mice showed that the CD19cherryluciferase transgenic mice are phenotypically normal with no underlying pathology as confirmed by necropsy and histologic analysis. Confocal microscopy followed by in vivo imaging of transgenic animals demonstrated that, as expected, expression of the cherryluciferase fusion protein is under the control of the CD19 locus regulatory elements and that the high sensitivity of bioluminescence imaging allows for the noninvasive quantification of luciferase expression in the secondary lymphoid organs. One of the more flexible aspects of the model is retained in the fact that the CD19 Cherryluciferase mouse can be crossed with most other models that spontaneously develop lymphoma representing a unique opportunity to study B-cell trafficking, in vivo measurement of tumor burden over the entire spectrum of the disease in individual animal and or response to therapy. To demonstrate its application double transgenic mice were produced by breeding CD19CherryLuciferase heterozygous transgenic animals with a Burkitt Lymphoma mouse model (Engel P et al Immunity 1995). A bioluminescent signal in double transgenic affected animals allowed tracking of B-cell lymphoma growth during a 4 week time period and evaluation of the response of an established B-cell lymphoma to a drug therapy (dexamethasone injected intraperitoneally at a dose of 4mg/kg). Relapse and accelerated tumor repopulation in a 5 day time period followed the rescue attempt. The possibility of sequential measurements of tumor growth as a function of an intervention allows essentially real time assessment and identification of micro- and macro-metastases in the living animals. At present there are no other models that allow in vivo imaging of spontaneously generated lymphoma and the mouse model described here is intended to be used to hasten translational studies of novel agents in lymphoma, with the intent that understanding the relevant pharmacology prior to clinical study will hasten successful development in clinical studies.