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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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Simultaneously inducing mutations and tracking mutated cells with a PB transposon system.(A) PB[mut-RFP] couples RFP expression with ectopic expression of a downstream gene or partial gene transcript via the IRES (brown box). CS (purple box) prevents unwanted RFP expression from transgene concatamers. (B) PB[mut-RFP] mobilization in HEK 293 cells induces RFP-positive clones that colocalize with myc-tagged proteins ectopically expressed by the mutator transposon. (C) Transgenic lines carrying two to 200 copies of PB[mut-RFP] were generated. (D) Significant PB transposition-induced embryonic lethality was observed when high-copy lines were crossed to Act-PBase. (E) Low-copy PB[mut-RFP] lines crossed to Act-PBase mobilized the mutator transposon as determined by PCR with primers directed against sequences flanking the transposon. A 212-bp excision product was specifically detected in double transgenics but not in single transgenic littermates. A 700-bp unexcised product was observed in all PB[mut-RFP] mice. (F) Shown here in the dorsal skin of a PB[mut-RFP]2a;Act-PBase mouse, RFP-positive foci can be detected ex vivo over background signal. Units, photons·s−1·sr−1·ìM−1. (G) RFP-marked tumor formation (arrowhead) can be visualized ex vivo as seen in the upper panels showing a PB[mut-RFP]2b;Act-PBase mouse. In the lower panels, a stomach tumor dissected from a PB[mut-RFP]7;Act-PBase mouse is strongly RFP-positive. Units, photons·s−1·sr−1·µM−1.
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pone-0026650-g003: Simultaneously inducing mutations and tracking mutated cells with a PB transposon system.(A) PB[mut-RFP] couples RFP expression with ectopic expression of a downstream gene or partial gene transcript via the IRES (brown box). CS (purple box) prevents unwanted RFP expression from transgene concatamers. (B) PB[mut-RFP] mobilization in HEK 293 cells induces RFP-positive clones that colocalize with myc-tagged proteins ectopically expressed by the mutator transposon. (C) Transgenic lines carrying two to 200 copies of PB[mut-RFP] were generated. (D) Significant PB transposition-induced embryonic lethality was observed when high-copy lines were crossed to Act-PBase. (E) Low-copy PB[mut-RFP] lines crossed to Act-PBase mobilized the mutator transposon as determined by PCR with primers directed against sequences flanking the transposon. A 212-bp excision product was specifically detected in double transgenics but not in single transgenic littermates. A 700-bp unexcised product was observed in all PB[mut-RFP] mice. (F) Shown here in the dorsal skin of a PB[mut-RFP]2a;Act-PBase mouse, RFP-positive foci can be detected ex vivo over background signal. Units, photons·s−1·sr−1·ìM−1. (G) RFP-marked tumor formation (arrowhead) can be visualized ex vivo as seen in the upper panels showing a PB[mut-RFP]2b;Act-PBase mouse. In the lower panels, a stomach tumor dissected from a PB[mut-RFP]7;Act-PBase mouse is strongly RFP-positive. Units, photons·s−1·sr−1·µM−1.

Mentions: In somatic mutagenesis, the ability to track mutated cells via the same transgenic line used to induce mutations opens up many experimental possibilities. We thus added new elements in our PB mutator transposon to label the mutant cells. The RFP coding sequence and an internal ribosome entry site (IRES) were inserted between the Actin promoter and myc-SD, which contains start codons in all three reading frames followed by a splice donor, of PB[mut] (Figure 3A, PB[mut-RFP]). Following insertions into introns or 5′ regulatory regions, production of bicistronic pre-mRNA containing RFP, IRES, the engineered myc-SD exon, and downstream endogenous intron and exon sequences can be initiated by the Actin promoter. After splicing of the myc-SD exon to downstream endogenous splice acceptors, the bicistronic transcripts enables the expression of RFP and the ectopic expression of endogenous proteins (Figure 3A). When we tested this mutator in cultured cells, the activated RFP marker was co-expressed in cells that ectopically expressed downstream endogenous proteins that incorporated the engineered myc-SD exon (Figure 3B). In order to ensure that PB[mut-RFP] does not express RFP from a transgenic concatamer, a concatamer silencer (CS) was inserted outside of the transposon (Figure 3A). The CS contains a splice acceptor followed by stop codons in all three reading frames to terminate transcripts initiated by the Actin promoter from transgene concatamers. To prevent alternative splicing–mediated exclusion of the CS splice acceptor, a sequence forming a pre-tRNA-like (ΔAC) structure was cloned after the stop codons. This substrate is efficiently recognized and cleaved by RNase P ensuring that pre-mRNA which contain the CS do not form mature transcripts [35].


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)

Simultaneously inducing mutations and tracking mutated cells with a PB transposon system.(A) PB[mut-RFP] couples RFP expression with ectopic expression of a downstream gene or partial gene transcript via the IRES (brown box). CS (purple box) prevents unwanted RFP expression from transgene concatamers. (B) PB[mut-RFP] mobilization in HEK 293 cells induces RFP-positive clones that colocalize with myc-tagged proteins ectopically expressed by the mutator transposon. (C) Transgenic lines carrying two to 200 copies of PB[mut-RFP] were generated. (D) Significant PB transposition-induced embryonic lethality was observed when high-copy lines were crossed to Act-PBase. (E) Low-copy PB[mut-RFP] lines crossed to Act-PBase mobilized the mutator transposon as determined by PCR with primers directed against sequences flanking the transposon. A 212-bp excision product was specifically detected in double transgenics but not in single transgenic littermates. A 700-bp unexcised product was observed in all PB[mut-RFP] mice. (F) Shown here in the dorsal skin of a PB[mut-RFP]2a;Act-PBase mouse, RFP-positive foci can be detected ex vivo over background signal. Units, photons·s−1·sr−1·ìM−1. (G) RFP-marked tumor formation (arrowhead) can be visualized ex vivo as seen in the upper panels showing a PB[mut-RFP]2b;Act-PBase mouse. In the lower panels, a stomach tumor dissected from a PB[mut-RFP]7;Act-PBase mouse is strongly RFP-positive. Units, photons·s−1·sr−1·µM−1.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026650-g003: Simultaneously inducing mutations and tracking mutated cells with a PB transposon system.(A) PB[mut-RFP] couples RFP expression with ectopic expression of a downstream gene or partial gene transcript via the IRES (brown box). CS (purple box) prevents unwanted RFP expression from transgene concatamers. (B) PB[mut-RFP] mobilization in HEK 293 cells induces RFP-positive clones that colocalize with myc-tagged proteins ectopically expressed by the mutator transposon. (C) Transgenic lines carrying two to 200 copies of PB[mut-RFP] were generated. (D) Significant PB transposition-induced embryonic lethality was observed when high-copy lines were crossed to Act-PBase. (E) Low-copy PB[mut-RFP] lines crossed to Act-PBase mobilized the mutator transposon as determined by PCR with primers directed against sequences flanking the transposon. A 212-bp excision product was specifically detected in double transgenics but not in single transgenic littermates. A 700-bp unexcised product was observed in all PB[mut-RFP] mice. (F) Shown here in the dorsal skin of a PB[mut-RFP]2a;Act-PBase mouse, RFP-positive foci can be detected ex vivo over background signal. Units, photons·s−1·sr−1·ìM−1. (G) RFP-marked tumor formation (arrowhead) can be visualized ex vivo as seen in the upper panels showing a PB[mut-RFP]2b;Act-PBase mouse. In the lower panels, a stomach tumor dissected from a PB[mut-RFP]7;Act-PBase mouse is strongly RFP-positive. Units, photons·s−1·sr−1·µM−1.
Mentions: In somatic mutagenesis, the ability to track mutated cells via the same transgenic line used to induce mutations opens up many experimental possibilities. We thus added new elements in our PB mutator transposon to label the mutant cells. The RFP coding sequence and an internal ribosome entry site (IRES) were inserted between the Actin promoter and myc-SD, which contains start codons in all three reading frames followed by a splice donor, of PB[mut] (Figure 3A, PB[mut-RFP]). Following insertions into introns or 5′ regulatory regions, production of bicistronic pre-mRNA containing RFP, IRES, the engineered myc-SD exon, and downstream endogenous intron and exon sequences can be initiated by the Actin promoter. After splicing of the myc-SD exon to downstream endogenous splice acceptors, the bicistronic transcripts enables the expression of RFP and the ectopic expression of endogenous proteins (Figure 3A). When we tested this mutator in cultured cells, the activated RFP marker was co-expressed in cells that ectopically expressed downstream endogenous proteins that incorporated the engineered myc-SD exon (Figure 3B). In order to ensure that PB[mut-RFP] does not express RFP from a transgenic concatamer, a concatamer silencer (CS) was inserted outside of the transposon (Figure 3A). The CS contains a splice acceptor followed by stop codons in all three reading frames to terminate transcripts initiated by the Actin promoter from transgene concatamers. To prevent alternative splicing–mediated exclusion of the CS splice acceptor, a sequence forming a pre-tRNA-like (ΔAC) structure was cloned after the stop codons. This substrate is efficiently recognized and cleaved by RNase P ensuring that pre-mRNA which contain the CS do not form mature transcripts [35].

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