Heat shock-inducible Cre/Lox approaches to induce diverse types of tumors and hyperplasia in transgenic zebrafish

Abstract

RAS family members are among the most frequently mutated oncogenes in human cancers. Given the utility of zebrafish in both chemical and genetic screens, developing RAS-induced cancer models will make large-scale screens possible to understand further the molecular mechanisms underlying malignancy. We developed a heat shock-inducible Cre/Lox-mediated transgenic approach in which activated human kRASG12D can be conditionally induced within transgenic animals by heat shock treatment. Specifically, double transgenic fish Tg(B-actin-LoxP-EGFP-LoxP-kRASG12D; hsp70-Cre) developed four types of tumors and hyperplasia after heat shock of whole zebrafish embryos, including rhabdomyosarcoma, myeloproliferative disorder, intestinal hyperplasia, and malignant peripheral nerve sheath tumor. Using ex vivo heat shock and transplantation of whole kidney marrow cells from double transgenic animals, we were able to generate specifically kRASG12D-induced myeloproliferative disorder in recipient fish. This heat shock-inducible recombination approach allowed for the generation of multiple types of RAS-induced tumors and hyperplasia without characterizing tissue-specific promoters. Moreover, these tumors and hyperplasia closely resemble human diseases at both the morphologic and molecular levels.myeloproliferative disorder ͉ RAS ͉ rhabdomyosarcoma ͉ intestine ͉ malignant peripheral nerve sheath tumor R AS genes encode a family of 21-kDa proteins that switch between inactive GDP-bound (RAS-GDP) and active GTPbound (RAS-GTP) conformations. Once in its activated GTPbound form, RAS interacts with downstream effectors to modulate diverse cellular responses, including proliferation, differentiation, and survival (1). Point mutations within RAS family members often occur at codon 12, 13, or 61 (2), which abolish RAS-GTP hydrolysis and lead to constitutive activation of downstream signaling pathways. These activating mutations are common in human malignancies; for example, Ͼ90% of pancreatic adenocarcinomas, 50% of colorectal cancers, 25-50% of lung cancers, 5-35% of rhabdomyosarcomas (RMS), and 25-50% of myeloid leukemia have mutational activation of RAS family members. Among the three different human RAS genes (H-, N-,

Co-injection strategies to modify radiation sensitivity and tumor initiation in transgenic Zebrafish

“…To capitalize on the powerful developmental and genetic tools available in zebrafish, researchers have created numerous zebrafish tumor models (Langenau et al, 2003Yang et al, 2004;Berghmans et al, 2005;Patton et al, 2005;Haramis et al, 2006;Sabaawy et al, 2006;Le et al, 2007). The ultimate goal of the majority of this work is to identify pathways that modify disease states.…”

A Proteoliposome Containing Apolipoprotein A-I Mutant (V156K) Enhances Rapid Tumor Regression Activity of Human Origin Oncolytic Adenovirus in Tumor-Bearing Zebrafish and Mice

“…2, a larger number of hematoxylin-stained cells was detected at the tumor site of DEN-treated zebrafish compared to that in control zebrafish. In addition, tumor sections showed less eosin-staining efficiency than normal tissue, suggesting that rapidly dividing cells, such as cancer cells, are less prone to staining, as reported by Le et al (2007). The DHE staining to visualize ROS showed that normal muscle exhibited a stronger red intensity due to eosin staining and fewer nuclei compared with those in tumor tissue, which had a larger number nuclei of increased size with weaker red staining intensity (Fig.…”

Functional characterization of lmo2‐Cre transgenic zebrafish

“…To generate the ␤-actin-loxPDsRed-loxP-EGFP construct, a 2.7-kb zebrafish ␤-actin promoter was excised from ␤-actin-loxP-EGFP-loxPkRASG12D plasmid (Le et al, 2007) by BamH I and Xho I, and blunted with Klenow fragment of E. coli DNA polymerase I., then cloned into the blunt-ended Xba I site of loxP-DsRed-SV40-loxP-PBSK-I-sceI plasmid (M. Deng et al, unpublished data). Finally, EGFP-SV40 fragment was excised from pCS2 ϩ -EGFP by Cla I and Kpn I and ligated into the same sites of ␤-actin-loxP-DsRed-loxP-PBSK-IsceI plasmid.…”

“…On the other hand, a heat-shock promoter-driven Tg(HSP70:EGFP-Cre) transgenic line was established and demonstrated to be able to mediate recombination when mated with a test line Tg (CMV:loxH-STOP-loxP-EGFP) after heat shock induction at 42°C (Thummel et al, 2005). Recently, Look's and Zon's groups reported that heat-shock promoter-driven Cre transgenic fish is able to induce DNA recombination in zebrafish adult, resulting in overexpression of oncogenic c-Myc or kRASG12D in the developing lymphoid cells and entire animal, respectively, and the subsequent development of T-cell acute lymphoblastic leukemia and hyperplasia in multiple tissues or tumors such as myeloproliferative disorder and rhabdomyosarcoma (Feng et al, 2007;Le et al, 2007). In spite of this progress, it remains unknown whether Cre recombinase is functional when expressed in a tissue-and/or cell subtype-specific manner.…”