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Rapid target gene validation in complex cancer mouse models using re-derived embryonic stem cells.

Huijbers IJ, Bin Ali R, Pritchard C, Cozijnsen M, Kwon MC, Proost N, Song JY, de Vries H, Badhai J, Sutherland K, Krimpenfort P, Michalak EM, Jonkers J, Berns A - EMBO Mol Med (2014)

Bottom Line: In our newly developed approach for the fast generation of tumor cohorts we have overcome this obstacle, as exemplified for three GEMMs; two lung cancer models and one mesothelioma model.Three elements are central for this system; (i) The efficient derivation of authentic Embryonic Stem Cells (ESCs) from established GEMMs, (ii) the routine introduction of transgenes of choice in these GEMM-ESCs by Flp recombinase-mediated integration and (iii) the direct use of the chimeric animals in tumor cohorts.As proof-of-principle, we demonstrate that MycL1 is a key driver gene in Small Cell Lung Cancer.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Genetics, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

ABSTRACT
Human cancers modeled in Genetically Engineered Mouse Models (GEMMs) can provide important mechanistic insights into the molecular basis of tumor development and enable testing of new intervention strategies. The inherent complexity of these models, with often multiple modified tumor suppressor genes and oncogenes, has hampered their use as preclinical models for validating cancer genes and drug targets. In our newly developed approach for the fast generation of tumor cohorts we have overcome this obstacle, as exemplified for three GEMMs; two lung cancer models and one mesothelioma model. Three elements are central for this system; (i) The efficient derivation of authentic Embryonic Stem Cells (ESCs) from established GEMMs, (ii) the routine introduction of transgenes of choice in these GEMM-ESCs by Flp recombinase-mediated integration and (iii) the direct use of the chimeric animals in tumor cohorts. By applying stringent quality controls, the GEMM-ESC approach proofs to be a reliable and effective method to speed up cancer gene assessment and target validation. As proof-of-principle, we demonstrate that MycL1 is a key driver gene in Small Cell Lung Cancer.

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Genomic stability of targeted GEMM-ESC clones.Comparison of chimeric contribution between the parental Rb1F/F;Trp53F/F ESC clone 1.5 and three Col1a1-frt targeted derivatives. Correct targeting was confirmed by Southern blot analysis using a 3′ probe in the Col1a1 locus (supplementary Fig S4A and B). Two Col1a1-frt targeted clones, i.e. 1.5_1A10 and 1.5_1B1, provided good and germline-competent chimeras (supplementary Table S1). One chimera from the Rb1F/F;Trp53F/F ESC clone 1.5_1B1 was backcrossed twice to the original strain and ESC were re-derived, i.e. clone 1.5_1B1 re-derived 4 (Table 1). This ESC clone resulted in improved chimeras compared to the parental clones.  male,  female,  n.d.Parts of whole representation of genetic aberrations observed in GEMM-ESCs cultured in 2i medium and subjected to either gene targeting, Flp-in integration and subcloning (supplementary Table S4). Last box represent the genetic aberrations observed in ESCs cultured under classic culture conditions as reported by Liang et al, 2008.Summary of CNVs observed in Rb1F/F;Trp53F/F ESC clones as detected by aCGH. Two Col1a1-frt targeted clones acquired four independent CNVs. Some CNVs can be transmitted via the germ line as CNV-4.1 was maintained after backcrossing twice to the original strain, see ESC clone 1.5_1B1 re-derived 4. A detailed description of all CNVs is provided in supplementary Table S3.
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fig03: Genomic stability of targeted GEMM-ESC clones.Comparison of chimeric contribution between the parental Rb1F/F;Trp53F/F ESC clone 1.5 and three Col1a1-frt targeted derivatives. Correct targeting was confirmed by Southern blot analysis using a 3′ probe in the Col1a1 locus (supplementary Fig S4A and B). Two Col1a1-frt targeted clones, i.e. 1.5_1A10 and 1.5_1B1, provided good and germline-competent chimeras (supplementary Table S1). One chimera from the Rb1F/F;Trp53F/F ESC clone 1.5_1B1 was backcrossed twice to the original strain and ESC were re-derived, i.e. clone 1.5_1B1 re-derived 4 (Table 1). This ESC clone resulted in improved chimeras compared to the parental clones. male, female, n.d.Parts of whole representation of genetic aberrations observed in GEMM-ESCs cultured in 2i medium and subjected to either gene targeting, Flp-in integration and subcloning (supplementary Table S4). Last box represent the genetic aberrations observed in ESCs cultured under classic culture conditions as reported by Liang et al, 2008.Summary of CNVs observed in Rb1F/F;Trp53F/F ESC clones as detected by aCGH. Two Col1a1-frt targeted clones acquired four independent CNVs. Some CNVs can be transmitted via the germ line as CNV-4.1 was maintained after backcrossing twice to the original strain, see ESC clone 1.5_1B1 re-derived 4. A detailed description of all CNVs is provided in supplementary Table S3.

Mentions: The second step in the GEMM-ESC approach involves targeting of GEMM-ESCs with a Flp-in module just after the 3′UTR of the Col1a1 locus (Beard et al, 2006). This module, named Col1a1-frt, serves as a docking site for introduction of transgene-coding plasmids by Flp recombinase-mediated integration. We choose this system as it is successfully applied by others (Zhu et al, 2009; Yilmaz et al, 2012), allows for transgene induction in multiple somatic cell types (Carey et al, 2010) and is compatible with a vector system for doxycycline-regulated, fluorescence-linked shRNAs (McJunkin et al, 2011; Premsrirut et al, 2011; Dow et al, 2012). Targetings were performed under the new 2i culture conditions; colonies were screened by PCR and correctly targeted clones were confirmed by Southern blotting (supplementary Fig S4A and B). For all three genotypes, i.e. Rb1F/F;Trp53F/F, Nf2F/F;Trp53F/F;Cdkn2a*/* and KrasLSL-G12D, similar targeting efficiencies of ˜35% were achieved (supplementary Table S2). These efficiencies were comparable to that of the wild-type 129/Ola ESC clone IB10 (36% under 2i culture conditions). The Col1a1-frt targeted GEMM-ESC clones were subsequently injected into morulae or blastocysts to produce chimeric mice. Out of 11 clones injected, three failed to produce chimeras. All other clones produced germline-competent chimeras (Fig 3A, supplementary Fig S5A and B, Table S1).


Rapid target gene validation in complex cancer mouse models using re-derived embryonic stem cells.

Huijbers IJ, Bin Ali R, Pritchard C, Cozijnsen M, Kwon MC, Proost N, Song JY, de Vries H, Badhai J, Sutherland K, Krimpenfort P, Michalak EM, Jonkers J, Berns A - EMBO Mol Med (2014)

Genomic stability of targeted GEMM-ESC clones.Comparison of chimeric contribution between the parental Rb1F/F;Trp53F/F ESC clone 1.5 and three Col1a1-frt targeted derivatives. Correct targeting was confirmed by Southern blot analysis using a 3′ probe in the Col1a1 locus (supplementary Fig S4A and B). Two Col1a1-frt targeted clones, i.e. 1.5_1A10 and 1.5_1B1, provided good and germline-competent chimeras (supplementary Table S1). One chimera from the Rb1F/F;Trp53F/F ESC clone 1.5_1B1 was backcrossed twice to the original strain and ESC were re-derived, i.e. clone 1.5_1B1 re-derived 4 (Table 1). This ESC clone resulted in improved chimeras compared to the parental clones.  male,  female,  n.d.Parts of whole representation of genetic aberrations observed in GEMM-ESCs cultured in 2i medium and subjected to either gene targeting, Flp-in integration and subcloning (supplementary Table S4). Last box represent the genetic aberrations observed in ESCs cultured under classic culture conditions as reported by Liang et al, 2008.Summary of CNVs observed in Rb1F/F;Trp53F/F ESC clones as detected by aCGH. Two Col1a1-frt targeted clones acquired four independent CNVs. Some CNVs can be transmitted via the germ line as CNV-4.1 was maintained after backcrossing twice to the original strain, see ESC clone 1.5_1B1 re-derived 4. A detailed description of all CNVs is provided in supplementary Table S3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3927956&req=5

fig03: Genomic stability of targeted GEMM-ESC clones.Comparison of chimeric contribution between the parental Rb1F/F;Trp53F/F ESC clone 1.5 and three Col1a1-frt targeted derivatives. Correct targeting was confirmed by Southern blot analysis using a 3′ probe in the Col1a1 locus (supplementary Fig S4A and B). Two Col1a1-frt targeted clones, i.e. 1.5_1A10 and 1.5_1B1, provided good and germline-competent chimeras (supplementary Table S1). One chimera from the Rb1F/F;Trp53F/F ESC clone 1.5_1B1 was backcrossed twice to the original strain and ESC were re-derived, i.e. clone 1.5_1B1 re-derived 4 (Table 1). This ESC clone resulted in improved chimeras compared to the parental clones. male, female, n.d.Parts of whole representation of genetic aberrations observed in GEMM-ESCs cultured in 2i medium and subjected to either gene targeting, Flp-in integration and subcloning (supplementary Table S4). Last box represent the genetic aberrations observed in ESCs cultured under classic culture conditions as reported by Liang et al, 2008.Summary of CNVs observed in Rb1F/F;Trp53F/F ESC clones as detected by aCGH. Two Col1a1-frt targeted clones acquired four independent CNVs. Some CNVs can be transmitted via the germ line as CNV-4.1 was maintained after backcrossing twice to the original strain, see ESC clone 1.5_1B1 re-derived 4. A detailed description of all CNVs is provided in supplementary Table S3.
Mentions: The second step in the GEMM-ESC approach involves targeting of GEMM-ESCs with a Flp-in module just after the 3′UTR of the Col1a1 locus (Beard et al, 2006). This module, named Col1a1-frt, serves as a docking site for introduction of transgene-coding plasmids by Flp recombinase-mediated integration. We choose this system as it is successfully applied by others (Zhu et al, 2009; Yilmaz et al, 2012), allows for transgene induction in multiple somatic cell types (Carey et al, 2010) and is compatible with a vector system for doxycycline-regulated, fluorescence-linked shRNAs (McJunkin et al, 2011; Premsrirut et al, 2011; Dow et al, 2012). Targetings were performed under the new 2i culture conditions; colonies were screened by PCR and correctly targeted clones were confirmed by Southern blotting (supplementary Fig S4A and B). For all three genotypes, i.e. Rb1F/F;Trp53F/F, Nf2F/F;Trp53F/F;Cdkn2a*/* and KrasLSL-G12D, similar targeting efficiencies of ˜35% were achieved (supplementary Table S2). These efficiencies were comparable to that of the wild-type 129/Ola ESC clone IB10 (36% under 2i culture conditions). The Col1a1-frt targeted GEMM-ESC clones were subsequently injected into morulae or blastocysts to produce chimeric mice. Out of 11 clones injected, three failed to produce chimeras. All other clones produced germline-competent chimeras (Fig 3A, supplementary Fig S5A and B, Table S1).

Bottom Line: In our newly developed approach for the fast generation of tumor cohorts we have overcome this obstacle, as exemplified for three GEMMs; two lung cancer models and one mesothelioma model.Three elements are central for this system; (i) The efficient derivation of authentic Embryonic Stem Cells (ESCs) from established GEMMs, (ii) the routine introduction of transgenes of choice in these GEMM-ESCs by Flp recombinase-mediated integration and (iii) the direct use of the chimeric animals in tumor cohorts.As proof-of-principle, we demonstrate that MycL1 is a key driver gene in Small Cell Lung Cancer.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Genetics, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

ABSTRACT
Human cancers modeled in Genetically Engineered Mouse Models (GEMMs) can provide important mechanistic insights into the molecular basis of tumor development and enable testing of new intervention strategies. The inherent complexity of these models, with often multiple modified tumor suppressor genes and oncogenes, has hampered their use as preclinical models for validating cancer genes and drug targets. In our newly developed approach for the fast generation of tumor cohorts we have overcome this obstacle, as exemplified for three GEMMs; two lung cancer models and one mesothelioma model. Three elements are central for this system; (i) The efficient derivation of authentic Embryonic Stem Cells (ESCs) from established GEMMs, (ii) the routine introduction of transgenes of choice in these GEMM-ESCs by Flp recombinase-mediated integration and (iii) the direct use of the chimeric animals in tumor cohorts. By applying stringent quality controls, the GEMM-ESC approach proofs to be a reliable and effective method to speed up cancer gene assessment and target validation. As proof-of-principle, we demonstrate that MycL1 is a key driver gene in Small Cell Lung Cancer.

Show MeSH
Related in: MedlinePlus