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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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Efficiency of the GEMM-ESC approach.Schematic representation of the GEMM-ESC approach including the performance of the individual steps. The approach is divided in two phases: a resource phase and an experimental phase. The resource phase includes ESC's derivation and targeting with the Col1a1-frt vector, performed once per GEMM and takes ˜6 months, including the necessary quality controls. The experimental phase is mainly focused on introducing a transgene-coding plasmid in a validated GEMM-ESC clone using the Flp-in method that allows for consecutive manipulations and takes ˜4 months to obtain a chimeric cohort. Alternatively, GEMM-ESC clones are also suitable for direct targeting of a specific gene or the introduction of mutant alleles using gene editing (arrows with dotted lines). The experimental phase also includes the option to follow an F1 route as almost all GEMM-ESC clones showed germline transmission (GLT). In practice, we advise that for each model (i) multiple Col1a1-frt targeted GEMM-ESC clones are screened for their ability to efficiently generate high quality chimeras, (ii) two of the best-performing clones are selected for the Flp-in procedure, and (iii) at least two transgene-coding GEMM-ESC clones are used to generate cohorts. The final clones should originate from different Col1a1-frt targeted parental clones to minimize the chance of miss-interpreting phenotypes due to possible unwanted genetic alterations introduced by long-term culture. The selection of best-performing Col1a1-frt targeted GEMM-ESC clones is crucial for the efficiency to later generate experimental cohorts as the number of chimeras born per injected embryo is likely to decline after additional manipulations and propagation in culture.
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fig05: Efficiency of the GEMM-ESC approach.Schematic representation of the GEMM-ESC approach including the performance of the individual steps. The approach is divided in two phases: a resource phase and an experimental phase. The resource phase includes ESC's derivation and targeting with the Col1a1-frt vector, performed once per GEMM and takes ˜6 months, including the necessary quality controls. The experimental phase is mainly focused on introducing a transgene-coding plasmid in a validated GEMM-ESC clone using the Flp-in method that allows for consecutive manipulations and takes ˜4 months to obtain a chimeric cohort. Alternatively, GEMM-ESC clones are also suitable for direct targeting of a specific gene or the introduction of mutant alleles using gene editing (arrows with dotted lines). The experimental phase also includes the option to follow an F1 route as almost all GEMM-ESC clones showed germline transmission (GLT). In practice, we advise that for each model (i) multiple Col1a1-frt targeted GEMM-ESC clones are screened for their ability to efficiently generate high quality chimeras, (ii) two of the best-performing clones are selected for the Flp-in procedure, and (iii) at least two transgene-coding GEMM-ESC clones are used to generate cohorts. The final clones should originate from different Col1a1-frt targeted parental clones to minimize the chance of miss-interpreting phenotypes due to possible unwanted genetic alterations introduced by long-term culture. The selection of best-performing Col1a1-frt targeted GEMM-ESC clones is crucial for the efficiency to later generate experimental cohorts as the number of chimeras born per injected embryo is likely to decline after additional manipulations and propagation in culture.

Mentions: The GEMM-ESC procedure as presented here, can be divided in two separate phases: a resource phase and an experimental phase (Fig 5). The resource phase, starting from the selection of the original GEMM until cryogenic storage of quality controlled Col1a1-frt targeted GEMM-ESC clones is fairly laborious, taking up to 6 months. The success rate is high and independent of the strain background. This can be largely attributed to the optimized ESC culture conditions, which not only simplify procedures but also allow for better quality ESCs as compared to previous protocols. The 2i culture protocol also enables derivation of ESCs from mouse strains that were previously considered refractory (Ying et al, 2008; Reinholdt et al, 2012). In this study, we derived 47 ESC clones from 364 embryos, representing 13% derivation efficiency. Although this seems low, most of our ESC derivation attempts were successful. The derivation efficiency could be further increased by expanding all early ESC clones instead of selecting them on the basis of their morphology and growth rate (Table 1).


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)

Efficiency of the GEMM-ESC approach.Schematic representation of the GEMM-ESC approach including the performance of the individual steps. The approach is divided in two phases: a resource phase and an experimental phase. The resource phase includes ESC's derivation and targeting with the Col1a1-frt vector, performed once per GEMM and takes ˜6 months, including the necessary quality controls. The experimental phase is mainly focused on introducing a transgene-coding plasmid in a validated GEMM-ESC clone using the Flp-in method that allows for consecutive manipulations and takes ˜4 months to obtain a chimeric cohort. Alternatively, GEMM-ESC clones are also suitable for direct targeting of a specific gene or the introduction of mutant alleles using gene editing (arrows with dotted lines). The experimental phase also includes the option to follow an F1 route as almost all GEMM-ESC clones showed germline transmission (GLT). In practice, we advise that for each model (i) multiple Col1a1-frt targeted GEMM-ESC clones are screened for their ability to efficiently generate high quality chimeras, (ii) two of the best-performing clones are selected for the Flp-in procedure, and (iii) at least two transgene-coding GEMM-ESC clones are used to generate cohorts. The final clones should originate from different Col1a1-frt targeted parental clones to minimize the chance of miss-interpreting phenotypes due to possible unwanted genetic alterations introduced by long-term culture. The selection of best-performing Col1a1-frt targeted GEMM-ESC clones is crucial for the efficiency to later generate experimental cohorts as the number of chimeras born per injected embryo is likely to decline after additional manipulations and propagation in culture.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig05: Efficiency of the GEMM-ESC approach.Schematic representation of the GEMM-ESC approach including the performance of the individual steps. The approach is divided in two phases: a resource phase and an experimental phase. The resource phase includes ESC's derivation and targeting with the Col1a1-frt vector, performed once per GEMM and takes ˜6 months, including the necessary quality controls. The experimental phase is mainly focused on introducing a transgene-coding plasmid in a validated GEMM-ESC clone using the Flp-in method that allows for consecutive manipulations and takes ˜4 months to obtain a chimeric cohort. Alternatively, GEMM-ESC clones are also suitable for direct targeting of a specific gene or the introduction of mutant alleles using gene editing (arrows with dotted lines). The experimental phase also includes the option to follow an F1 route as almost all GEMM-ESC clones showed germline transmission (GLT). In practice, we advise that for each model (i) multiple Col1a1-frt targeted GEMM-ESC clones are screened for their ability to efficiently generate high quality chimeras, (ii) two of the best-performing clones are selected for the Flp-in procedure, and (iii) at least two transgene-coding GEMM-ESC clones are used to generate cohorts. The final clones should originate from different Col1a1-frt targeted parental clones to minimize the chance of miss-interpreting phenotypes due to possible unwanted genetic alterations introduced by long-term culture. The selection of best-performing Col1a1-frt targeted GEMM-ESC clones is crucial for the efficiency to later generate experimental cohorts as the number of chimeras born per injected embryo is likely to decline after additional manipulations and propagation in culture.
Mentions: The GEMM-ESC procedure as presented here, can be divided in two separate phases: a resource phase and an experimental phase (Fig 5). The resource phase, starting from the selection of the original GEMM until cryogenic storage of quality controlled Col1a1-frt targeted GEMM-ESC clones is fairly laborious, taking up to 6 months. The success rate is high and independent of the strain background. This can be largely attributed to the optimized ESC culture conditions, which not only simplify procedures but also allow for better quality ESCs as compared to previous protocols. The 2i culture protocol also enables derivation of ESCs from mouse strains that were previously considered refractory (Ying et al, 2008; Reinholdt et al, 2012). In this study, we derived 47 ESC clones from 364 embryos, representing 13% derivation efficiency. Although this seems low, most of our ESC derivation attempts were successful. The derivation efficiency could be further increased by expanding all early ESC clones instead of selecting them on the basis of their morphology and growth rate (Table 1).

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