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A Cre-conditional MYCN-driven neuroblastoma mouse model as an improved tool for preclinical studies.

Althoff K, Beckers A, Bell E, Nortmeyer M, Thor T, Sprüssel A, Lindner S, De Preter K, Florin A, Heukamp LC, Klein-Hitpass L, Astrahantseff K, Kumps C, Speleman F, Eggert A, Westermann F, Schramm A, Schulte JH - Oncogene (2014)

Bottom Line: The availability of valid preclinical in vivo models is a prerequisite to develop novel targeted therapies.Gene set enrichment analyses demonstrated significant correlation with MYC-associated expression patterns.Treatment of a cell line established from a tumor derived from a LSL-MYCN;Dbh-iCre mouse with JQ1 or MLN8237 reduced cell viability and demonstrated oncogene addiction to MYCN.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pediatric Oncology and Hematology, University Children's Hospital Essen, Essen, Germany [2] German Cancer Consortium (DKTK), Partner Site Essen/Duesseldorf, Hufelandstr, Germany.

ABSTRACT
Neuroblastoma, a childhood cancer that originates from neural crest-derived cells, is the most common deadly solid tumor of infancy. Amplification of the MYCN oncogene, which occurs in approximately 20-25% of human neuroblastomas, is the most prominent genetic marker of high-stage disease. The availability of valid preclinical in vivo models is a prerequisite to develop novel targeted therapies. We here report on the generation of transgenic mice with Cre-conditional induction of MYCN in dopamine β-hydroxylase-expressing cells, termed LSL-MYCN;Dbh-iCre. These mice develop neuroblastic tumors with an incidence of >75%, regardless of strain background. Molecular profiling of tumors revealed upregulation of the MYCN-dependent miR-17-92 cluster as well as expression of neuroblastoma marker genes, including tyrosine hydroxylase and the neural cell adhesion molecule 1. Gene set enrichment analyses demonstrated significant correlation with MYC-associated expression patterns. Array comparative genome hybridization showed that chromosomal aberrations in LSL-MYCN;Dbh-iCre tumors were syntenic to those observed in human neuroblastomas. Treatment of a cell line established from a tumor derived from a LSL-MYCN;Dbh-iCre mouse with JQ1 or MLN8237 reduced cell viability and demonstrated oncogene addiction to MYCN. Here we report establishment of the first Cre-conditional human MYCN-driven mouse model for neuroblastoma that closely recapitulates the human disease with respect to tumor localization, histology, marker expression and genomic make up. This mouse model is a valuable tool for further functional studies and to assess the effect of targeted therapies.

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Generation of transgenic LSL-MYCN mice. (a) Graphical representation of the ROSA26 locus with recombinase-mediated cassette exchange (RMCE) sites used to introduce the RMCE exchange vector containing the MYCN transgene. The Rosa26 locus is displayed before (top) and after (center) insertion of MYCN by RMCE, and after cre-recombinase-mediated removal of the transcription termination site 5′ to the MYCN allele (bottom). Localizations of primers used for genotyping (A1 and A2) and the PCR-based validation of floxing out the transcriptional site 5′ of the MYCN allele (B1 and B2) are displayed. Splice acceptor site (SA), polyadenylation signal (pA), internal ribosome entry site (IRES), chicken actin gene promotor (CAG), transcriptional STOP cassette made of the human Growth Hormone polyadenylation signal (hGHpA), human MYCN open reading frame (MYCN). (b) Representative genotyping PCR validating the MYCN knock-in allele in heterozygous and homozygous LSL-MYCN mice (primers used: A1 and A2); wild type (wt), heterozygous LSL-MYCN (+/−), homozygous LSL-MYCN (+/+). (c) Representative PCR validating absence or presence of the transgene inserted into the ROSA26 locus in wt, heterozygous (+/−) and homozygous (+/+) LSL-MYCN mice.
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fig1: Generation of transgenic LSL-MYCN mice. (a) Graphical representation of the ROSA26 locus with recombinase-mediated cassette exchange (RMCE) sites used to introduce the RMCE exchange vector containing the MYCN transgene. The Rosa26 locus is displayed before (top) and after (center) insertion of MYCN by RMCE, and after cre-recombinase-mediated removal of the transcription termination site 5′ to the MYCN allele (bottom). Localizations of primers used for genotyping (A1 and A2) and the PCR-based validation of floxing out the transcriptional site 5′ of the MYCN allele (B1 and B2) are displayed. Splice acceptor site (SA), polyadenylation signal (pA), internal ribosome entry site (IRES), chicken actin gene promotor (CAG), transcriptional STOP cassette made of the human Growth Hormone polyadenylation signal (hGHpA), human MYCN open reading frame (MYCN). (b) Representative genotyping PCR validating the MYCN knock-in allele in heterozygous and homozygous LSL-MYCN mice (primers used: A1 and A2); wild type (wt), heterozygous LSL-MYCN (+/−), homozygous LSL-MYCN (+/+). (c) Representative PCR validating absence or presence of the transgene inserted into the ROSA26 locus in wt, heterozygous (+/−) and homozygous (+/+) LSL-MYCN mice.

Mentions: LSL-MYCN transgenic mice were viable and fertile without obvious physiological or morphological phenotypes. Offspring resulting from breeding LSL-MYCN mice with wild-type mice were born according to the expected Mendelian ratio (data not shown). We next confirmed the molecular integration site and localization of the transgene. Representative examples of PCR analyses validating transgene integration in heterozygous and homozygous LSL-MYCN mice are shown in Figure 1b. PCR analyses validating the presence of a wild-type allele at the ROSA26 locus in wild-type and heterozygous LSL-MYCN mice are shown in Figure 1c.


A Cre-conditional MYCN-driven neuroblastoma mouse model as an improved tool for preclinical studies.

Althoff K, Beckers A, Bell E, Nortmeyer M, Thor T, Sprüssel A, Lindner S, De Preter K, Florin A, Heukamp LC, Klein-Hitpass L, Astrahantseff K, Kumps C, Speleman F, Eggert A, Westermann F, Schramm A, Schulte JH - Oncogene (2014)

Generation of transgenic LSL-MYCN mice. (a) Graphical representation of the ROSA26 locus with recombinase-mediated cassette exchange (RMCE) sites used to introduce the RMCE exchange vector containing the MYCN transgene. The Rosa26 locus is displayed before (top) and after (center) insertion of MYCN by RMCE, and after cre-recombinase-mediated removal of the transcription termination site 5′ to the MYCN allele (bottom). Localizations of primers used for genotyping (A1 and A2) and the PCR-based validation of floxing out the transcriptional site 5′ of the MYCN allele (B1 and B2) are displayed. Splice acceptor site (SA), polyadenylation signal (pA), internal ribosome entry site (IRES), chicken actin gene promotor (CAG), transcriptional STOP cassette made of the human Growth Hormone polyadenylation signal (hGHpA), human MYCN open reading frame (MYCN). (b) Representative genotyping PCR validating the MYCN knock-in allele in heterozygous and homozygous LSL-MYCN mice (primers used: A1 and A2); wild type (wt), heterozygous LSL-MYCN (+/−), homozygous LSL-MYCN (+/+). (c) Representative PCR validating absence or presence of the transgene inserted into the ROSA26 locus in wt, heterozygous (+/−) and homozygous (+/+) LSL-MYCN mice.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4487199&req=5

fig1: Generation of transgenic LSL-MYCN mice. (a) Graphical representation of the ROSA26 locus with recombinase-mediated cassette exchange (RMCE) sites used to introduce the RMCE exchange vector containing the MYCN transgene. The Rosa26 locus is displayed before (top) and after (center) insertion of MYCN by RMCE, and after cre-recombinase-mediated removal of the transcription termination site 5′ to the MYCN allele (bottom). Localizations of primers used for genotyping (A1 and A2) and the PCR-based validation of floxing out the transcriptional site 5′ of the MYCN allele (B1 and B2) are displayed. Splice acceptor site (SA), polyadenylation signal (pA), internal ribosome entry site (IRES), chicken actin gene promotor (CAG), transcriptional STOP cassette made of the human Growth Hormone polyadenylation signal (hGHpA), human MYCN open reading frame (MYCN). (b) Representative genotyping PCR validating the MYCN knock-in allele in heterozygous and homozygous LSL-MYCN mice (primers used: A1 and A2); wild type (wt), heterozygous LSL-MYCN (+/−), homozygous LSL-MYCN (+/+). (c) Representative PCR validating absence or presence of the transgene inserted into the ROSA26 locus in wt, heterozygous (+/−) and homozygous (+/+) LSL-MYCN mice.
Mentions: LSL-MYCN transgenic mice were viable and fertile without obvious physiological or morphological phenotypes. Offspring resulting from breeding LSL-MYCN mice with wild-type mice were born according to the expected Mendelian ratio (data not shown). We next confirmed the molecular integration site and localization of the transgene. Representative examples of PCR analyses validating transgene integration in heterozygous and homozygous LSL-MYCN mice are shown in Figure 1b. PCR analyses validating the presence of a wild-type allele at the ROSA26 locus in wild-type and heterozygous LSL-MYCN mice are shown in Figure 1c.

Bottom Line: The availability of valid preclinical in vivo models is a prerequisite to develop novel targeted therapies.Gene set enrichment analyses demonstrated significant correlation with MYC-associated expression patterns.Treatment of a cell line established from a tumor derived from a LSL-MYCN;Dbh-iCre mouse with JQ1 or MLN8237 reduced cell viability and demonstrated oncogene addiction to MYCN.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pediatric Oncology and Hematology, University Children's Hospital Essen, Essen, Germany [2] German Cancer Consortium (DKTK), Partner Site Essen/Duesseldorf, Hufelandstr, Germany.

ABSTRACT
Neuroblastoma, a childhood cancer that originates from neural crest-derived cells, is the most common deadly solid tumor of infancy. Amplification of the MYCN oncogene, which occurs in approximately 20-25% of human neuroblastomas, is the most prominent genetic marker of high-stage disease. The availability of valid preclinical in vivo models is a prerequisite to develop novel targeted therapies. We here report on the generation of transgenic mice with Cre-conditional induction of MYCN in dopamine β-hydroxylase-expressing cells, termed LSL-MYCN;Dbh-iCre. These mice develop neuroblastic tumors with an incidence of >75%, regardless of strain background. Molecular profiling of tumors revealed upregulation of the MYCN-dependent miR-17-92 cluster as well as expression of neuroblastoma marker genes, including tyrosine hydroxylase and the neural cell adhesion molecule 1. Gene set enrichment analyses demonstrated significant correlation with MYC-associated expression patterns. Array comparative genome hybridization showed that chromosomal aberrations in LSL-MYCN;Dbh-iCre tumors were syntenic to those observed in human neuroblastomas. Treatment of a cell line established from a tumor derived from a LSL-MYCN;Dbh-iCre mouse with JQ1 or MLN8237 reduced cell viability and demonstrated oncogene addiction to MYCN. Here we report establishment of the first Cre-conditional human MYCN-driven mouse model for neuroblastoma that closely recapitulates the human disease with respect to tumor localization, histology, marker expression and genomic make up. This mouse model is a valuable tool for further functional studies and to assess the effect of targeted therapies.

Show MeSH
Related in: MedlinePlus