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piggyBac transposon somatic mutagenesis with an activated reporter and tracker (PB-SMART) for genetic screens in mice.

Landrette SF, Cornett JC, Ni TK, Bosenberg MW, Xu T - PLoS ONE (2011)

Bottom Line: Somatic forward genetic screens have the power to interrogate thousands of genes in a single animal.Furthermore, locating mutant clones is a prerequisite for screening and analyzing most other somatic phenotypes.We demonstrate that PB-SMART is highly mutagenic, capable of tumor induction with low copy transposons, which facilitates the mapping and identification of causative insertions.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Yale University School of Medicine, Boyer Center for Molecular Medicine, Howard Hughes Medical Institute, New Haven, Connecticut, United States of America.

ABSTRACT
Somatic forward genetic screens have the power to interrogate thousands of genes in a single animal. Retroviral and transposon mutagenesis systems in mice have been designed and deployed in somatic tissues for surveying hematopoietic and solid tumor formation. In the context of cancer, the ability to visually mark mutant cells would present tremendous advantages for identifying tumor formation, monitoring tumor growth over time, and tracking tumor infiltrations and metastases into wild-type tissues. Furthermore, locating mutant clones is a prerequisite for screening and analyzing most other somatic phenotypes. For this purpose, we developed a system using the piggyBac (PB) transposon for somatic mutagenesis with an activated reporter and tracker, called PB-SMART. The PB-SMART mouse genetic screening system can simultaneously induce somatic mutations and mark mutated cells using bioluminescence or fluorescence. The marking of mutant cells enable analyses that are not possible with current somatic mutagenesis systems, such as tracking cell proliferation and tumor growth, detecting tumor cell infiltrations, and reporting tissue mutagenesis levels by a simple ex vivo visual readout. We demonstrate that PB-SMART is highly mutagenic, capable of tumor induction with low copy transposons, which facilitates the mapping and identification of causative insertions. We further integrated a conditional transposase with the PB-SMART system, permitting tissue-specific mutagenesis with a single cross to any available Cre line. Targeting the germline, the system could also be used to conduct F1 screens. With these features, PB-SMART provides an integrated platform for individual investigators to harness the power of somatic mutagenesis and phenotypic screens to decipher the genetic basis of mammalian biology and disease.

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Identification of driver oncogenes in solid tumors from Luc-PB[mut]7;Act-PBase mice.(A) Insertions (wide arrows) upstream of the M-isoform of Mitf were mapped from five kidney tumors. (B) Quantitative PCR reveals that Mitf transcripts are upregulated in all five kidney tumors (KT1, KT2, KT3, KT4, and KT5) compared to two wild-type kidneys (wt-K). (C) Kidney tumors possessed similar histological profiles, featuring packets of spindle-shaped epitheliod cells, indicating carcinoma (scale bar, 50 µm). (D) Histological analysis shows SRBC morphology (scale bar, 100 µm). (E) Insertions (wide arrows) in intron 7 or 8 of Gli2 were mapped from eleven SRBC tumors.
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pone-0026650-g002: Identification of driver oncogenes in solid tumors from Luc-PB[mut]7;Act-PBase mice.(A) Insertions (wide arrows) upstream of the M-isoform of Mitf were mapped from five kidney tumors. (B) Quantitative PCR reveals that Mitf transcripts are upregulated in all five kidney tumors (KT1, KT2, KT3, KT4, and KT5) compared to two wild-type kidneys (wt-K). (C) Kidney tumors possessed similar histological profiles, featuring packets of spindle-shaped epitheliod cells, indicating carcinoma (scale bar, 50 µm). (D) Histological analysis shows SRBC morphology (scale bar, 100 µm). (E) Insertions (wide arrows) in intron 7 or 8 of Gli2 were mapped from eleven SRBC tumors.

Mentions: In five kidney tumors, we identified common insertions in Mitf. The mutator transposons in all five tumors were inserted in the coding direction, upstream of the translational start of the M-isoform, suggesting overexpression of Mitf in these tumors (Figure 2A). Indeed, quantitative PCR revealed that Mitf was upregulated in these kidney tumors as compared to two wild-type kidney samples (Figure 2B). Histopathology of the kidney tumors revealed an expansion of nests of pleomorphic spindle and epithelioid cells suggestive of carcinoma (Figure 2C).


piggyBac transposon somatic mutagenesis with an activated reporter and tracker (PB-SMART) for genetic screens in mice.

Landrette SF, Cornett JC, Ni TK, Bosenberg MW, Xu T - PLoS ONE (2011)

Identification of driver oncogenes in solid tumors from Luc-PB[mut]7;Act-PBase mice.(A) Insertions (wide arrows) upstream of the M-isoform of Mitf were mapped from five kidney tumors. (B) Quantitative PCR reveals that Mitf transcripts are upregulated in all five kidney tumors (KT1, KT2, KT3, KT4, and KT5) compared to two wild-type kidneys (wt-K). (C) Kidney tumors possessed similar histological profiles, featuring packets of spindle-shaped epitheliod cells, indicating carcinoma (scale bar, 50 µm). (D) Histological analysis shows SRBC morphology (scale bar, 100 µm). (E) Insertions (wide arrows) in intron 7 or 8 of Gli2 were mapped from eleven SRBC tumors.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198810&req=5

pone-0026650-g002: Identification of driver oncogenes in solid tumors from Luc-PB[mut]7;Act-PBase mice.(A) Insertions (wide arrows) upstream of the M-isoform of Mitf were mapped from five kidney tumors. (B) Quantitative PCR reveals that Mitf transcripts are upregulated in all five kidney tumors (KT1, KT2, KT3, KT4, and KT5) compared to two wild-type kidneys (wt-K). (C) Kidney tumors possessed similar histological profiles, featuring packets of spindle-shaped epitheliod cells, indicating carcinoma (scale bar, 50 µm). (D) Histological analysis shows SRBC morphology (scale bar, 100 µm). (E) Insertions (wide arrows) in intron 7 or 8 of Gli2 were mapped from eleven SRBC tumors.
Mentions: In five kidney tumors, we identified common insertions in Mitf. The mutator transposons in all five tumors were inserted in the coding direction, upstream of the translational start of the M-isoform, suggesting overexpression of Mitf in these tumors (Figure 2A). Indeed, quantitative PCR revealed that Mitf was upregulated in these kidney tumors as compared to two wild-type kidney samples (Figure 2B). Histopathology of the kidney tumors revealed an expansion of nests of pleomorphic spindle and epithelioid cells suggestive of carcinoma (Figure 2C).

Bottom Line: Somatic forward genetic screens have the power to interrogate thousands of genes in a single animal.Furthermore, locating mutant clones is a prerequisite for screening and analyzing most other somatic phenotypes.We demonstrate that PB-SMART is highly mutagenic, capable of tumor induction with low copy transposons, which facilitates the mapping and identification of causative insertions.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Yale University School of Medicine, Boyer Center for Molecular Medicine, Howard Hughes Medical Institute, New Haven, Connecticut, United States of America.

ABSTRACT
Somatic forward genetic screens have the power to interrogate thousands of genes in a single animal. Retroviral and transposon mutagenesis systems in mice have been designed and deployed in somatic tissues for surveying hematopoietic and solid tumor formation. In the context of cancer, the ability to visually mark mutant cells would present tremendous advantages for identifying tumor formation, monitoring tumor growth over time, and tracking tumor infiltrations and metastases into wild-type tissues. Furthermore, locating mutant clones is a prerequisite for screening and analyzing most other somatic phenotypes. For this purpose, we developed a system using the piggyBac (PB) transposon for somatic mutagenesis with an activated reporter and tracker, called PB-SMART. The PB-SMART mouse genetic screening system can simultaneously induce somatic mutations and mark mutated cells using bioluminescence or fluorescence. The marking of mutant cells enable analyses that are not possible with current somatic mutagenesis systems, such as tracking cell proliferation and tumor growth, detecting tumor cell infiltrations, and reporting tissue mutagenesis levels by a simple ex vivo visual readout. We demonstrate that PB-SMART is highly mutagenic, capable of tumor induction with low copy transposons, which facilitates the mapping and identification of causative insertions. We further integrated a conditional transposase with the PB-SMART system, permitting tissue-specific mutagenesis with a single cross to any available Cre line. Targeting the germline, the system could also be used to conduct F1 screens. With these features, PB-SMART provides an integrated platform for individual investigators to harness the power of somatic mutagenesis and phenotypic screens to decipher the genetic basis of mammalian biology and disease.

Show MeSH
Related in: MedlinePlus