Number Of EGFP-positive Cells Research Articles

A minimal promoter region of Kit gene recapitulates mast cell differentiation in development, aging and inflammation

To follow mast cells (MCs) distribution during aging and inflammation, we characterized two transgenic mouse models in which the EGFP expression is controlled by 9 kb or 12 kb of Kit gene promoter, defined as p18 and p70, respectively. We detected EGFP-positive cells in the serosal surfaces of the peritoneum, pleuras and pericardium, mucosal cavities, and connective tissue of almost all organs including gonads of p70, but not of p18 mice. By FACS and immunofluorescence for FcεR1, Kit and β7-integrin, we found that these EGFP positive cells were MCs. In non-inflammatory conditions, a higher percentage of EGFP positive cells was found in juvenile with respect to adult serosal surfaces, but no differences between males and females at both developmental ages. We found, however, a striking difference in developing gonads, with low numbers of EGFP positive cells in fetal ovaries compared to age matched testes. Under inflammatory conditions caused by high fat diet (HFD), mice showed an increase in serosal EGFP positve cells. Altogether our results identify a regulatory region of the Kit gene, activated in MCs and that directing EGFP expression, can be employed to trace this immune cell type throughout the organism and in different animal conditions.

Krüppel-like factor 7 protects retinal ganglion cells and promotes functional preservation via activating the Akt pathway after retinal ischemia-reperfusion injury

ObjectiveThe purpose of this study is to investigate the effects of Krüppel-like factor 7 (KLF7) on retinal ganglion cells (RGCs) and retinal function after retinal ischemia-reperfusion (RIR) injury in mice. MethodsMale C57BL/6J mice were intravitreally injected with recombinant adeno-associated vectors (rAAV-KLF7-EGFP or rAAV-EGFP), and subsequently used to induce RIR injury. Retinal cryosections were used to access the efficacy of virus transfection, 1, 2, 3, and 4 weeks after rAAV-KLF7-EGFP transfer. RGCs survival rate was observed and quantified by immunofluorescent staining, 7 days after RIR injury. Meanwhile, electroretinogram (ERG) and optomotor response were used to evaluate the electrophysiological functions and visual acuity. Apoptosis was evaluated by TUNEL staining 1 day after RIR injury. Expression of KLF7, Akt, phospho-Akt, Bcl-2, and Bax were further detected by western blot to excavate the underlying mechanism. ResultsThe transfection efficiency of rAAV-KLF7-EGFP was increased in a time-dependent manner, and the number of EGFP-positive cells was increased significantly 3 weeks after rAAV-KLF7-EGFP transfer. RGCs survival rates, amplitudes of ERG a-, b-wave, Ops, PhNR, and visual acuity of mice were decreased after RIR injury. With the increase of light intensity, the amplitudes of scotopic ERG a- and b-wave were gradually increased while the incubation period was gradually shortened. RGCs survival rates, amplitudes of ERG a-, b-wave, Ops, PhNR, and visual acuity of mice were increased after rAAV-KLF7-EGFP transfer. The protein level of KLF7 was up-regulated after rAAV-KLF7-EGFP transfer. Up-regulation of KLF7 significantly inhibited cells apoptosis, increased phospho-Akt and Bcl-2 expression, and decreased Bax expression. There were no significant changes in Akt expression. ConclusionOverexpression of KLF7 can not only prevent the loss of RGCs, but also preserve the electrophysiological function. In addition, overexpression of KLF7 can ameliorate the retinal dysfunction after RIR injury, and ultimately improve the visual acuity of mice. The activation of Akt pathway and the suppression of the mitochondrial apoptotic pathway contribute to the neuroprotection of KLF7.

Collagen Microspheres: A Three-Dimensional Culture System for Notochordal Cells

IntroductionThe notochord plays a crucial role in the formation and patterning of axial skeleton. Notochordal cells (NCCs) have drawn increasing attentions from developmental biology, stem cells research and tissue engineering in these years as they are important in development and maintenance of NP. However, it is difficult to maintain their survival and phenotype in 2D culture for long term, making it difficult to understand their fate and functional maintenance during intervertebral disc development. This project is to develop a better culture system that is able to maintain the survival and phenotypes of NCCs. Materials and MethodsFoxa2mNE–Cre/Z/EG heterozygous embryos were developed and NCCs could be identified EGFP signal. NCC isolated with or without FACS-sorting, notochord segments at the anterior, trunk and posterior regions, and whole notochord pulled out from embryo were microencapsulated respectively in type I collagen microspheres and cultured for up to 1 month before characterization for survival and phenotype maintenance. ResultsEGFP signal of sorted cells could be maintained after 4 weeks. The EGFP positive cells in collagen microspheres showed colocalization with foxa2 and brachyury, which were considered as markers of NCCs, indicated that they were still NCCs. However, areas with these merged were very rare. EGFP signal of unsorted cells could be maintained and clusters of EGFP positive cells were showed during 4-weeks culture. When the notochord segments were cultured in collagen microspheres, clusters of round EGFP positive cells co-expressing with major NCC markers were found after 1 month, suggesting that NCC phenotype was maintained. Moreover, the number of EGFP positive cells was increased during culture. In the other hand, when the notochord was cultured in collagen microsphere, it started pinching and the pinching region seemed growing at day 28. ConclusionType I collagen microspheres present a potential culture system for increasing cell number with EGFP positive signal for at least 4 weeks. NCCs entrapped in type I collagen microspheres could maintained the NCCs phenotype. Comparing with FACS-sorted NCCs, there was increasing number of NCCs in unsorted group, segment culture and whole notochord culture, suggesting that the presence of niche cells, extracellular matrix, and native configuration of notochord may be important to support NCCs growth and phenotype maintenance. AcknowledgmentsThis work was supported by AoE (AoE/M-04/04), TBRS (T12–708/12-N) and AOSPN (SRN_2011_14).Disclosures: None

Phytohemagglutinin facilitates the aggregation of blastomere pairs from Day 5 donor embryos with Day 4 host embryos for chimeric bovine embryo multiplication

Multiplication of bovine embryos by the production of aggregation chimeras is based on the concept that few blastomeres of a donor embryo form the inner cell mass (ICM) and thus the embryo proper, whereas cells of a host embryo preferentially contribute to the trophectoderm (TE), the progenitor cells of the embryonic part of the placenta. We aggregated two fluorescent blastomeres from enhanced green fluorescent protein (eGFP) transgenic Day 5 morulae with two Day 4 embryos that did not complete their first cleavage until 27 hours after IVF and tested the effect of phytohemagglutinin-L (PHA) on chimeric embryo formation. The resulting blastocysts were characterized by differential staining of cell lineages using the TE-specific factor CDX2 and confocal laser scanning microscopy to facilitate the precise localization of eGFP-positive cells. The proportions of blastocyst development of sandwich aggregates with (n = 99) and without PHA (n = 46) were 85.9% and 54.3% (P < 0.05), respectively. Epifluorescence microscopy showed that the proportion of blastocysts with eGFP-positive cells in the ICM was higher in the PHA group than in the no-PHA group (40% vs. 16%; P < 0.05). Confocal laser scanning microscopy revealed that the total cell numbers of blastocysts from the PHA group of aggregation chimeras (n = 17; 207.8 ± 67.3 [mean ± standard deviation]) were higher (P < 0.05) than those of embryos without ZP and exposed to PHA (n = 30; 159.6 ± 42.2) and of handling control embryos (n = 19; 176.9 ± 53.3). The same was true for ICM cell counts (56.5 ± 22.0 vs. 37.7 ± 14.2 and 38.7 ± 12.4) and TE cell counts (151.2 ± 58.0 vs. 121.9 ± 37.4 and 138.3 ± 53.0), whereas the ICM/total cell number ratio was not different between the groups. Of the 17 chimeric blastocysts analyzed by confocal laser scanning microscopy, nine had eGFP-positive cells (three of them in the ICM, three in the TE, and three in both lineages). When integration in the ICM occurred, the number of eGFP-positive cells in this compartment was 8.3 ± 2.3 (mean ± standard error of the mean). We conclude that PHA is advantageous for the formation of aggregation chimeras, but the approach tested in the present study with only two donor blastomeres and two host embryos did not result in multiplication of genetically valuable donor embryos.

Expression of GDNF Throughout Rat Brain After Intranasal Delivery of hGDNF Plasmid Nanoparticles

Glial cell line‐derived neurotrophic factor (GDNF) gene therapy is a promising approach for CNS disorders such as Parkinson's disease (PD). We are examining intranasal administration of PEG‐CK30 DNA nanoparticles (NPs) encoding hGDNF, developed by Copernicus Therapeutics, Inc. We have shown that intranasal NPs incorporating hGDNF DNA (pGDNF), or a hGDNF‐eGFP fusion plasmid (pUGG), transfect brain cells in vivo, and that intranasal pGDNF NPs protect substantia nigra (SN) dopamine neurons in a rat model of PD.Here, we examined transfection in rat brain 7 days after intranasal delivery of these NPs. ELISA showed increased GDNF levels throughout the brain of rats given pGDNF relative to saline controls. Immunohistochemistry (IHC) revealed that the number of eGFP positive cells was significantly higher in rats given intranasal pUGG NPs compared to background fluorescence in controls, with the highest numbers in midbrain. Double label IHC showed that most eGFP positive cells were immediately adjacent to capillary endothelial cells staining for RECA‐1, and they were often contiguous to GFAP positive astrocytic endfeet enwrapping capillaries. When eGFP cells were observed adjacent to dopamine neurons in SN, or NeuN labeled cells in any region, these neurons were also found close to a capillary. This pattern suggests that pericytes are transfected after intranasal administration of DNA NPs, consistent with distribution by perivascular transport. These results confirm delivery and transfection of cells in rat brain after intranasal administration of Copernicus' DNA NPs and support use of intranasal hGDNF NPs as a non‐invasive gene therapy for PD.

192 IDENTIFICATION AND CHARACTERIZATION OF Oct4-EGFP EXPRESSING CELLS IN TRANSGENIC PIG TESTIS

We have produced germ line transgenic pigs carrying the entire 18-kb genomic sequence of the murine Oct4 gene fused to the enhanced green fluorescent protein (EGFP) cDNA (OG2 construct; Nowak-Imialek et al., 2011 Stem Cells Dev.). Expression of the EGFP reporter construct is confined to germ line cells, the inner cell mass, and trophectoderm of blastocysts, and testicular germ cells, including putative spermatogonial stem cells (SSC). SSC are unique among stem cells because they can both self-renew and differentiate into spermatozoa. In-depth knowledge on porcine SSC has been hampered by the inability to isolate these cells from the complex cell population of the testis. In the Oct4-EGFP transgenic mouse, SSC are the only adult stem cells that express Oct4. Fluorescence microscopy of testicular tissue isolated from transgenic piglets revealed minimum numbers of EGFP-positive cells, whereas testicular tissue isolated from adult transgenic boars contained a high amount of EGFP fluorescent cells. Northern blot analysis confirmed stronger EGFP expression in the testis of adult transgenic pigs than in the testis from transgenic piglets. Time course and the signal intensity of EGFP expression in Oct4-EGFP testis paralleled mRNA expression of the endogenous Oct4 gene. Here, we used adult Oct4-EGFP transgenic pigs as a model for fluorescence-activated cell sorting (FACS)-based isolation of EGFP-expressing cells from testes. To obtain a single-cell suspension, the testes were enzymatically dissociated using two digestion steps. Thereafter, FACS based on EGFP expression was successfully used to purify specific testicular cell populations. Two cell populations, i.e. EGFP+ (14%) and EGFP– (45%) could be isolated. Subsequently, qualitative PCR analyses were performed on EGFP+, EGFP–, and unsorted cell populations using marker genes specific for pluripotency and undifferentiated germ cells (OCT4, FGFR3, UTF1, PGP9.5, GFRα1, CD90, SALL4), differentiating germ cells (c-KIT), meiosis (BOLL), spermatids (PRM2), and somatic cells (VIM, LHCGR). All of the genes, including OCT4, UTF1, FGFR3, PGP9.5, CD90, SALL4, and GFRα1 were expressed at least 3-fold and up to 12-fold greater in the EGFP-positive population. Vimentin, which is mainly expressed in Sertoli cells and LHCGR, which is mainly expressed in Leydig cells, were expressed in unsorted and EGFP– cell populations and at very low level in EGFP+ cells. Moreover, expression of the c-KIT and PRM2 markers were detected also in EGFP+ cell population, indicating that these cells contain also differentiating spermatogonia. To explore the characteristics of the Oct4-EGFP expressing cells in greater detail, localization in the porcine testis sections and analysis of co-expression with germ cell markers using immunohistochemistry is currently underway.

Expanding or Restricting the Target Site Repertoire of Zinc-finger Nucleases: The Inter-domain Linker as a Major Determinant of Target Site Selectivity

Precise manipulations of complex genomes by zinc-finger nucleases (ZFNs) depend on site-specific DNA cleavage, which requires two ZFN subunits to bind to two target half-sites separated by a spacer of 6 base pairs (bp). ZFN subunits consist of a specific DNA-binding domain and a nonspecific cleavage domain, connected by a short inter-domain linker. In this study, we conducted a systematic analysis of 11 candidate-based linkers using episomal and chromosomal targets in two human cell lines. We achieved gene targeting in up to 20% of transfected cells and identified linker variants that enforce DNA cleavage at narrowly defined spacer lengths and linkers that expand the repertoire of potential target sites. For instance, a nine amino acid (aa) linker induced efficient gene conversion at chromosomal sites with 7- or 16-bp spacers, whereas 4-aa linkers had activity optima at 5- and 6-bp spacers. Notably, single aa substitutions in the 4-aa linker affected the ZFN activity significantly, and both gene conversion and ZFN-associated toxicity depended on the linker/spacer combination and the cell type. In summary, both sequence and length of the inter-domain linker determine ZFN activity and target-site specificity, and are therefore important parameters to account for when designing ZFNs for genome editing.

Optimal conditions for freezing CHO‐S and HEK293‐EBNA cell lines: Influence of Me2SO, freeze density, and PEI‐mediated transfection on revitalization and growth of cells, and expression of recombinant protein

To avoid the time consuming, labor intensive seed-train expansion and to improve production reliability and consistency, portions of bulk cryopreserved cells from the same cultivation can be utilized as inocula or alternatively may be used to undertake transient transfections for large-scale bioreactor production. In this study, the conditions for large-scale freezing in cryobags were optimized utilizing a design of experiment approach. We showed that relatively high density of 30-40 x 10(6) cells/mL and relatively low Me(2)SO concentrations of 5-6% in the freezing media are optimal to freeze HEK293-EBNA and CHO-S cells in a controlled manner in order to achieve high viable cell recovery and growth post-thawing. The immediate transfer of freshly thawed cells into culture medium resulted in better cell growth compared to cells that were centrifuged in order to remove Me(2)SO. This was the case as long as the residual Me(2)SO did not exceed 0.2-0.3%. The best time to perform transient 25 kDa polyethylenimine-mediated transfection of pCEP4-EGFP plasmid into freshly thawed, one-step inoculated cells is after 72-96 h in culture. At this time point, the numbers of EGFP-positive cells in the freshly thawed culture mimic perfectly that of cells grown continuously. Finally, our data showed that it is possible to freeze transiently polyethyleneimine-transfected HEK293-EBNA cells and maintain growth rate and expression of recombinant protein following thawing. The optimal time point for freezing cells was 4 h after transfection.

Developmental rate and allocation of transgenic cells in rabbit chimeric embryos

The objective of this study was to compare developmental capacity of rabbit chimeric embryos and the allocation of the EGFP gene expression to the embryoblast (ICM) or embryonic shield. We produced chimeric embryos (TR< >N) by synchronous transfer of two or three blastomeres at the 16-cell stage from transgenic (TR) into normal host embryos (N) at the same stage. In the control group, two to three non-transgenic blastomeres were used to produce chimeric embryos. The TR embryos were produced by microinjection of EGFP into both pronuclei of fertilized rabbit eggs. The developmental rate and allocation of EGFP-positive cells of the reconstructed chimeric embryos was controlled at blastocyst (96 h PC) and embryonic shield (day 6) stage. All chimeric embryos (120/120, 100%) developed up to blastocyst stage. Using fluorescent microscope, we detected green signal (EGFP expression). In 90 chimeric (TR< >N) embryos (75%). Average total number of cells in chimeric embryos at blastocyst stage was 175+/-13.10, of which 58+/-2.76 cells were found in the ICM area. The number of EGFP-positive cells in the ICM area was 24+/-5.02 (35%). After the transfer of 50 chimeric rabbit embryos at the 16-cell stage, 20 embryos (40%) were flushed from five recipients on day 6 of pregnancy, of which five embryos (25%) were EGFP positive at the embryonic shield stage. Our results demonstrate that transgenic blastomeres in synchronous chimeric embryos reconstructed from TR embryos have an ability to develop and colonize ICM and embryonic shield area.

Development of transplantable genetically modified corneal epithelial cell sheets for gene therapy

The purpose of this study was to establish a method for the fabrication of exogenous gene-transferred, transplantable corneal epithelial cell sheets. Corneo-limbal epithelial cells collected from USA eye bank eyes were transduced with an EGFP-expressing lentiviral vector at differential MOI. Multi-layered corneal epithelial cell sheets were fabricated by co-cultivation of transduced cells and mitomycin C-treated 3T3 feeder layers on temperature-responsive culture dishes. These cultured epithelial cells could be harvested as intact sheets by simply lowering the temperature. The number of EGFP-positive cells was increased as the MOI raised, and at an MOI of 100, nearly 100% of the superficial cells showed strong EGFP expression. Histological analysis revealed that EGFP was expressed in all layers of the cell sheet of which cell source was transduced with the lentiviral vector at an MOI of 100. Immunofluorescence data showed that p63 was also expressed in the basal layer of the same cell sheet. These results suggest that this technique will likely be applicable to ex vivo gene therapies for various corneal disorders.

Identification of PML-RARa Target Genes Using Microarray and ChIP-on-Chip Analysis.

PML-RARa is very toxic to early myeloid cell lines that express neutrophil elastase. As a consequence, myeloid cell lines that stably express PML-RARa must undergo significant adaptations to avoid toxicity (eg. loss of expression of neutrophil elastase (Lane and Ley, MCB, 2005). To identify the direct target genes of PML-RARa, we decided to bypass the issue of adaptation by profiling naive U937 cells that transiently expressed PML-RARa. To define “immediate-early” expression changes caused by PML-RARa, we harvested U937 cells electroporated with expression vectors containing eGFP-PML-RARa (known to possess all the measurable functional properties of PML-RARa) vs. eGFP alone, at the earliest time point when adequate numbers of eGFP positive cells could be collected via high speed cell sorting (6 hours). RNA obtained from these cells was subjected to linear amplification and hybridized with Affymetrix U133 Plus 2.0 arrays. Probesets that demonstrated the largest differences in expression between cells transfected with eGFP vs. eGFP-PML-RARa were PML and RARa themselves (15-fold increase for PML, 9-fold for RARa), providing an important validation for the experimental system. Differentially expressed genes were selected by three filters for further study: (i) ANOVA statistic (P < 0.05); (ii) “present” call and >150 average expression units in at least one sample; and (iii) Reproducible (n=2) >2-fold differences between eGFP-PML-RARa vs. eGFP vector transfected cells. Only 280 probesets (out of 54,613 total) met all of these criteria. Among them, 37 genes were involved in signal transduction, 15 in cell proliferation and differentiation, 7 in apoptosis, 14 in cell cycle, 15 in transport, 27 in protein metabolism and modification, 13 in oncogenesis, and 13 in immunity and defense (Panther classification system). 15/15 genes selected from a variety of categories were further validated with Q-PCR in duplicate using replicate samples, and all differences were statistically significant (p<0.05). Q-PCR validated genes that were up-regulated at 6 hours by eGFP-PML-RARa included CD36 (196% increase over eGFP vector control), LATS2 (204%), Sox4 (395%), COPEB (325%), HoxA5 (294%), IFITM3 (139%), AIF1 (197%), and PLD1 (315%). Q-PCR validated genes that were down-regulated by eGFP-PML-RARa at 6 hours included BTG2 (60% the level of eGFP vector control), KLF7(52%), CDKN1A (p21)(39%), Pim1 (58%), SLC23A2 (36%), HoxA13 (51%), and PDCD1LG1 (42%). Among these genes, Sox4, Pim1, BTG2, and PDCD1LG1 have previously been implicated in cancer pathogenesis by retroviral insertional mutagenesis (http://RTCGD.ncifcrf.gov ). To identify promoters that were directly in contact with the eGFP-PML-RARa fusion protein, we performed “ChIP-on-chip” analysis using chromatin immunoprecipitation coupled with a NimbleGen promoter tiling array containing 15 50-mer probes (~100 bp spacing) between positions −1500 and +500 of the transcription initiation sites of a curated subset of 24,275 known human genes (HG17, Build 35). By subtracting non-specific hybridization signals from the eGFP control chip, we identified and validated several promoter regions as direct binding targets of eGFP-PML-RARa, including HoxA13 and CDKN1A (p21). This experimental system provides a new approach for the identification of the direct transcriptional targets of PML-RARa, which may lead to new insights into APL pathogenesis and treatment.

Focal Adhesion Kinase Signaling Regulates Cardiogenesis of Embryonic Stem Cells

The signaling steps that induce cardiac differentiation in embryonic stem (ES) cells are incompletely understood. We examined the effect of adhesion signaling including Src and focal adhesion kinase (FAK) on cardiogenesis in mouse ES cells using alpha-myosin heavy chain promoter-driven enhanced green fluorescent protein or luciferase as reporters. Cardiac transcription factors including Nkx2.5 and Tbx5 mRNA were first expressed at day 4 in hanging drop embryoid bodies, and adhesion of embryoid bodies to surfaces at or before that day strongly inhibited differentiation of ES cells to cardiomyocytes. Since adhesion signaling could suppress cardiogenesis through Src kinases, embryoid bodies were exposed to the small molecule PP2, known as a Src family kinase inhibitor. PP2 during embryoid body adhesion dramatically increased cardiomyocyte differentiation and decreased mRNA expression of neuronal cellular adhesion molecule and alpha-fetoprotein, neuroectodermal, and endodermal markers, respectively. Surprisingly, although there was an interaction between Src and FAK in cardiogenesis, the procardiogenic effect of PP2 appeared incompletely explained by Src kinase inhibition, since another Src family kinase inhibitor, SU6656, failed to induce cardiogenesis. Instead, PP2 specifically inhibited adhesion-induced FAK phosphorylation. In ES cells stably expressing FAK-related nonkinase, which functions as a dominant negative FAK, cell migration from embryoid bodies was inhibited, whereas alpha-myosin heavy chain expression and myosin-stained cardiomyocytes were increased, suggesting that reducing cell motility may contribute to cardiogenesis. These data indicate that FAK is a key regulator of cardiogenesis in mouse ES cells and that FAK signaling within embryoid bodies can direct stem cell lineage commitment.

Ectopic Fibroblast Growth Factor Receptor 3 (FGFR3) Expression Mediated by Isotype Switch Recombination.

FGFR3 is disregulated in approximately 15% of cases of multiple myeloma (MM) occurring by chromosomal translocation in the Igh switch region which results in the juxtaposition of FGFR3 next to the regulatory elements of the IgH locus, causing its ectopic expression in plasma cells. The acquisition of FGFR3 activating mutations in some tumors indicate a role for FGFR3 in tumor progression. In order to study its role in MM progression we developed a strain of transgenic mice in which the expression of the activated form of FGFR3 is mediated by isotype switch recombination by replacing the IgH gamma1 costant region with an FGFR3-IRES-EGFP cassette in a BAC that covers the entire murine IgH locus. We predict that in the transgenic mice B cells that undergo switch recombination to g1 on the productive allele will also undergo switch recombination to g1 at the transgenic locus and express FGFR3 and EGFP. To evaluate FGFR3 expression in vitro, we collected splenocytes from our transgenic mice and induced their proliferation and differentiation to plasma cells (mainly IgG1) using LPS and IL4. After four days of stimulation, the average number of EGFP positive cells went from 0.2% to 80%. We then analyzed FGFR3 expression by RT PCR, Northern and Western blot and detected LPS/IL4 inducible FGFR3 expression in plasma cells mediated mainly by the Igamma1 promoter and in a subset of them by the Vh promoter. We then tested if forced FGFR3 expression in B cells would affect their proliferation rate and ability to differentiate in vitro by MTT assay, cell cycle analysis and PI staining on LPS/IL4 stimulated splenocytes at different time points. With this system, we did not observe any FGFR3 mediated alteration of splenocytes growth parameters when compared to wild type controls. Similarly, in vivo no monoclonal gammopathy has been observed by serum protein electrophoresis and all the transgenic mice remaine tumor free at 1.5 years. Although we have not been able to demonstrate a tumorigenic role for FGFR3 in switched plasmacells we were able to obtaine regulated FGFR3 expression in them. It is possible that FGFR3 expression alone is not sufficient to induce plasma cells transformation and that FGFR3 has a dispensable role in tumor initiation, but that it could still play a role in tumor progression. Consistently, FGFR3 expression is lost in about 25% of multiple myeloma wih a t(4;14). To address this hypotesis we are currently crossing these mice with MMSET (the other gene disregulated in the t(4;14)) transgenic mice to investigate a possible collaborative role between these two genes in the development of MM.