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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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Tissue-specific PB-SMART.(A) Activation of the conditional LSL-PBase enables tissue-specific mutagenesis by crossing transgenic mice to available Cre lines. (B) Luc-PB[mut]7;LSL-PBase;Ksp-Cre transgenics (red bars) undergo PB mutagenesis specifically in the seminal vesicles and kidneys, reported by luciferase signal. Control mouse: Luc-PB[mut]7;LSL-PBase (blue bars). Units, photons·s−1·cm−2·sr−1. (C) PB mutagenesis throughout the skin of Luc-PB[mut]7;LSL-PBase;K14-Cre mice (red bar) is reported by luciferase signal. Control mouse: Luc-PB[mut]7;K14-Cre (blue bar). Units, photons·s−1·cm−2·sr−1. (D) Tyr-CreER conditionally activates BrafV600E and PB mutagenesis specifically in melanocytes of quadruple transgenic mice (Luc-PB[mut]7;LSL-PBase;BrafCA;Tyr-CreER, red bars). The expansion of this cell population at three, nine, and eleven months (left, middle and right, respectively) is tracked ex vivo by luciferase signal. Control mouse: Luc-PB[mut]7;BrafCA;Tyr-CreER (blue bars). Units, photons·s−1·cm−2·sr−1.
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pone-0026650-g005: Tissue-specific PB-SMART.(A) Activation of the conditional LSL-PBase enables tissue-specific mutagenesis by crossing transgenic mice to available Cre lines. (B) Luc-PB[mut]7;LSL-PBase;Ksp-Cre transgenics (red bars) undergo PB mutagenesis specifically in the seminal vesicles and kidneys, reported by luciferase signal. Control mouse: Luc-PB[mut]7;LSL-PBase (blue bars). Units, photons·s−1·cm−2·sr−1. (C) PB mutagenesis throughout the skin of Luc-PB[mut]7;LSL-PBase;K14-Cre mice (red bar) is reported by luciferase signal. Control mouse: Luc-PB[mut]7;K14-Cre (blue bar). Units, photons·s−1·cm−2·sr−1. (D) Tyr-CreER conditionally activates BrafV600E and PB mutagenesis specifically in melanocytes of quadruple transgenic mice (Luc-PB[mut]7;LSL-PBase;BrafCA;Tyr-CreER, red bars). The expansion of this cell population at three, nine, and eleven months (left, middle and right, respectively) is tracked ex vivo by luciferase signal. Control mouse: Luc-PB[mut]7;BrafCA;Tyr-CreER (blue bars). Units, photons·s−1·cm−2·sr−1.

Mentions: We further expanded the PB-SMART system for tissue-specific genetic screens. To allow the system to be readily adopted for screens in many types of tissues and cells, we generated transgenic mice in which PBase or PBaseER can be activated by tissue-specific Cre by inserting a Stop-pA sequence flanked by loxP sites between the Actin promoter and the transposase coding sequence (Figure 5A, LSL-PBase, LSL-PBaseER). When these double transgenics were crossed into lines expressing Cre in the kidney and genitourinary tract or the skin epithelium [36], [37], correct targeting of mutagenesis to these tissues was confirmed by the luciferase reporter (Figures 5B and 5C).


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)

Tissue-specific PB-SMART.(A) Activation of the conditional LSL-PBase enables tissue-specific mutagenesis by crossing transgenic mice to available Cre lines. (B) Luc-PB[mut]7;LSL-PBase;Ksp-Cre transgenics (red bars) undergo PB mutagenesis specifically in the seminal vesicles and kidneys, reported by luciferase signal. Control mouse: Luc-PB[mut]7;LSL-PBase (blue bars). Units, photons·s−1·cm−2·sr−1. (C) PB mutagenesis throughout the skin of Luc-PB[mut]7;LSL-PBase;K14-Cre mice (red bar) is reported by luciferase signal. Control mouse: Luc-PB[mut]7;K14-Cre (blue bar). Units, photons·s−1·cm−2·sr−1. (D) Tyr-CreER conditionally activates BrafV600E and PB mutagenesis specifically in melanocytes of quadruple transgenic mice (Luc-PB[mut]7;LSL-PBase;BrafCA;Tyr-CreER, red bars). The expansion of this cell population at three, nine, and eleven months (left, middle and right, respectively) is tracked ex vivo by luciferase signal. Control mouse: Luc-PB[mut]7;BrafCA;Tyr-CreER (blue bars). Units, photons·s−1·cm−2·sr−1.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198810&req=5

pone-0026650-g005: Tissue-specific PB-SMART.(A) Activation of the conditional LSL-PBase enables tissue-specific mutagenesis by crossing transgenic mice to available Cre lines. (B) Luc-PB[mut]7;LSL-PBase;Ksp-Cre transgenics (red bars) undergo PB mutagenesis specifically in the seminal vesicles and kidneys, reported by luciferase signal. Control mouse: Luc-PB[mut]7;LSL-PBase (blue bars). Units, photons·s−1·cm−2·sr−1. (C) PB mutagenesis throughout the skin of Luc-PB[mut]7;LSL-PBase;K14-Cre mice (red bar) is reported by luciferase signal. Control mouse: Luc-PB[mut]7;K14-Cre (blue bar). Units, photons·s−1·cm−2·sr−1. (D) Tyr-CreER conditionally activates BrafV600E and PB mutagenesis specifically in melanocytes of quadruple transgenic mice (Luc-PB[mut]7;LSL-PBase;BrafCA;Tyr-CreER, red bars). The expansion of this cell population at three, nine, and eleven months (left, middle and right, respectively) is tracked ex vivo by luciferase signal. Control mouse: Luc-PB[mut]7;BrafCA;Tyr-CreER (blue bars). Units, photons·s−1·cm−2·sr−1.
Mentions: We further expanded the PB-SMART system for tissue-specific genetic screens. To allow the system to be readily adopted for screens in many types of tissues and cells, we generated transgenic mice in which PBase or PBaseER can be activated by tissue-specific Cre by inserting a Stop-pA sequence flanked by loxP sites between the Actin promoter and the transposase coding sequence (Figure 5A, LSL-PBase, LSL-PBaseER). When these double transgenics were crossed into lines expressing Cre in the kidney and genitourinary tract or the skin epithelium [36], [37], correct targeting of mutagenesis to these tissues was confirmed by the luciferase reporter (Figures 5B and 5C).

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