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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 reporting mutagenesis activity levels with a PB transposon system.(A) The Actin promoter (black pointed box) and myc-splice donor sequence (blue box) are engineered in the PB[mut] transposon to ectopically express downstream genes or partial transcripts. A splice acceptor and transcriptional termination sequences (pink box) truncate transcripts initiating upstream of the transposon insertion site. PB[mut] is inserted within an internal TTAA sequence of the luciferase gene to create Luc-PB[mut]. Transposase excises PB[mut] from the luciferase gene, restoring the luciferase expression and thus labeling cells. (B) By ex vivo luciferase imaging, PB mutagenesis occurs in Luc-PB[mut]7;Act-PBase mice (right) but not in single transgenic littermates (left and middle). Units, photons·s−1·cm−2·sr−1.(C) Quantitative PCR analysis of transposon excision in the Luc-PB[mut]7 line demonstrates a 30-fold increase in transposition activity between Act-PBase and Act-PBaseER (top). The difference in mutagenesis activity levels driven by Act-PBaseER (middle) and Act-PBase lines (right) can be differentiated by the luciferase PB reporter. Units, photons·s−1·cm−2·sr−1. (D) Tumor-free survival curves over the span of 50 weeks show that Luc-PB[mut]7;Act-PBase(Cre) mice succumb to tumors while Luc-PB[mut]7(Cre) and Act-PBase(Cre) littermates remain relatively tumor-free.
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pone-0026650-g001: Simultaneously inducing mutations and reporting mutagenesis activity levels with a PB transposon system.(A) The Actin promoter (black pointed box) and myc-splice donor sequence (blue box) are engineered in the PB[mut] transposon to ectopically express downstream genes or partial transcripts. A splice acceptor and transcriptional termination sequences (pink box) truncate transcripts initiating upstream of the transposon insertion site. PB[mut] is inserted within an internal TTAA sequence of the luciferase gene to create Luc-PB[mut]. Transposase excises PB[mut] from the luciferase gene, restoring the luciferase expression and thus labeling cells. (B) By ex vivo luciferase imaging, PB mutagenesis occurs in Luc-PB[mut]7;Act-PBase mice (right) but not in single transgenic littermates (left and middle). Units, photons·s−1·cm−2·sr−1.(C) Quantitative PCR analysis of transposon excision in the Luc-PB[mut]7 line demonstrates a 30-fold increase in transposition activity between Act-PBase and Act-PBaseER (top). The difference in mutagenesis activity levels driven by Act-PBaseER (middle) and Act-PBase lines (right) can be differentiated by the luciferase PB reporter. Units, photons·s−1·cm−2·sr−1. (D) Tumor-free survival curves over the span of 50 weeks show that Luc-PB[mut]7;Act-PBase(Cre) mice succumb to tumors while Luc-PB[mut]7(Cre) and Act-PBase(Cre) littermates remain relatively tumor-free.

Mentions: PB has previously been found to integrate at high frequency near or within genes [3]. To efficiently induce somatic mutations, we generated a PB mutator transposon (Figure 1A, PB[mut]) to induce ectopic gene expression in multiple genomic contexts. The CMV early enhancer/chicken β-actin promoter was cloned between the PB transposon arms to induce overexpression of genes downstream of a transposon insertion site. A myc epitope in all three reading frames and a splice donor were placed after the Actin promoter such that transcription and translation initiation can incorporate the myc tag sequence prior to splicing into endogenous exons. The CMV enhancer can also act as a bidirectional enhancer to upregulate genes. Additionally, in the event that PB[mut] inserts into an intron, an N-terminally truncated product can be ectopically induced. Insertions into introns can also produce C-terminally truncated gene products initiated by the endogenous promoter. A splice acceptor followed by a sequence with stop codons in all three frames and a poly(A) signal was added for this purpose. Expression of truncated genes may produce dominant active and dominant negative proteins. Thus, by design, the PB[mut] transposon can induce the ectopic expression of full-length or truncated endogenous genes in many different genomic contexts (Figure 1A). Indeed, PB[mut] can induce ectopic full-length and truncated gene expression in PB-SMART screens (see below).


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 reporting mutagenesis activity levels with a PB transposon system.(A) The Actin promoter (black pointed box) and myc-splice donor sequence (blue box) are engineered in the PB[mut] transposon to ectopically express downstream genes or partial transcripts. A splice acceptor and transcriptional termination sequences (pink box) truncate transcripts initiating upstream of the transposon insertion site. PB[mut] is inserted within an internal TTAA sequence of the luciferase gene to create Luc-PB[mut]. Transposase excises PB[mut] from the luciferase gene, restoring the luciferase expression and thus labeling cells. (B) By ex vivo luciferase imaging, PB mutagenesis occurs in Luc-PB[mut]7;Act-PBase mice (right) but not in single transgenic littermates (left and middle). Units, photons·s−1·cm−2·sr−1.(C) Quantitative PCR analysis of transposon excision in the Luc-PB[mut]7 line demonstrates a 30-fold increase in transposition activity between Act-PBase and Act-PBaseER (top). The difference in mutagenesis activity levels driven by Act-PBaseER (middle) and Act-PBase lines (right) can be differentiated by the luciferase PB reporter. Units, photons·s−1·cm−2·sr−1. (D) Tumor-free survival curves over the span of 50 weeks show that Luc-PB[mut]7;Act-PBase(Cre) mice succumb to tumors while Luc-PB[mut]7(Cre) and Act-PBase(Cre) littermates remain relatively tumor-free.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026650-g001: Simultaneously inducing mutations and reporting mutagenesis activity levels with a PB transposon system.(A) The Actin promoter (black pointed box) and myc-splice donor sequence (blue box) are engineered in the PB[mut] transposon to ectopically express downstream genes or partial transcripts. A splice acceptor and transcriptional termination sequences (pink box) truncate transcripts initiating upstream of the transposon insertion site. PB[mut] is inserted within an internal TTAA sequence of the luciferase gene to create Luc-PB[mut]. Transposase excises PB[mut] from the luciferase gene, restoring the luciferase expression and thus labeling cells. (B) By ex vivo luciferase imaging, PB mutagenesis occurs in Luc-PB[mut]7;Act-PBase mice (right) but not in single transgenic littermates (left and middle). Units, photons·s−1·cm−2·sr−1.(C) Quantitative PCR analysis of transposon excision in the Luc-PB[mut]7 line demonstrates a 30-fold increase in transposition activity between Act-PBase and Act-PBaseER (top). The difference in mutagenesis activity levels driven by Act-PBaseER (middle) and Act-PBase lines (right) can be differentiated by the luciferase PB reporter. Units, photons·s−1·cm−2·sr−1. (D) Tumor-free survival curves over the span of 50 weeks show that Luc-PB[mut]7;Act-PBase(Cre) mice succumb to tumors while Luc-PB[mut]7(Cre) and Act-PBase(Cre) littermates remain relatively tumor-free.
Mentions: PB has previously been found to integrate at high frequency near or within genes [3]. To efficiently induce somatic mutations, we generated a PB mutator transposon (Figure 1A, PB[mut]) to induce ectopic gene expression in multiple genomic contexts. The CMV early enhancer/chicken β-actin promoter was cloned between the PB transposon arms to induce overexpression of genes downstream of a transposon insertion site. A myc epitope in all three reading frames and a splice donor were placed after the Actin promoter such that transcription and translation initiation can incorporate the myc tag sequence prior to splicing into endogenous exons. The CMV enhancer can also act as a bidirectional enhancer to upregulate genes. Additionally, in the event that PB[mut] inserts into an intron, an N-terminally truncated product can be ectopically induced. Insertions into introns can also produce C-terminally truncated gene products initiated by the endogenous promoter. A splice acceptor followed by a sequence with stop codons in all three frames and a poly(A) signal was added for this purpose. Expression of truncated genes may produce dominant active and dominant negative proteins. Thus, by design, the PB[mut] transposon can induce the ectopic expression of full-length or truncated endogenous genes in many different genomic contexts (Figure 1A). Indeed, PB[mut] can induce ectopic full-length and truncated gene expression in PB-SMART screens (see below).

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