piggyBac Transposon Somatic Mutagenesis with an Activated Reporter and Tracker (PB-SMART) for Genetic Screens in Mice

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

Plag1 and Plagl2 are oncogenes that induce acute myeloid leukemia in cooperation with Cbfb-MYH11

Recurrent chromosomal rearrangements are associated with the development of acute myeloid leukemia (AML). The frequent inversion of chromosome 16 creates the CBFB-MYH11 fusion gene that encodes the fusion protein CBFbeta-SMMHC. This fusion protein inhibits the core-binding factor (CBF), resulting in a block of hematopoietic differentiation, and induces leukemia upon the acquisition of additional mutations. A recent genetic screen identified Plag1 and Plagl2 as CBF beta-SMMHC candidate cooperating proteins. In this study, we demonstrate that Plag1 and Plagl2 independently cooperate with CBF beta-SMMHC in vivo to efficiently trigger leukemia with short latency in the mouse. In addition, Plag1 and Plagl2 increased proliferation by inducing G1 to S transition that resulted in the expansion of hematopoietic progenitors and increased cell renewal in vitro. Finally, PLAG1 and PLAGL2 expression was increased in 20% of human AML samples. Interestingly, PLAGL2 was preferentially increased in samples with chromosome 16 inversion, suggesting that PLAG1 and PLAGL2 may also contribute to human AML. Overall, this study shows that Plag1 and Plagl2 are novel leukemia oncogenes that act by expanding hematopoietic progenitors expressing CbF beta-SMMHC

Characterization of Fluorescent Eye Markers for Mammalian Transgenic Studies

Genotyping mice by DNA based methods is both laborious and costly. As an alternative, we systematically examined fluorescent proteins expressed in the lens as transgenic markers for mice. A set of eye markers has been selected such that double and triple transgenic animals can be visually identified and that fluorescence intensity in the eyes can be used to distinguish heterozygous from homozygous mice. Taken together, these eye markers dramatically reduce the time and cost of genotyping transgenics and empower analysis of genetic interaction

Apoptosis in a MPTP Parkinson's disease mouse model

The purpose of this project was to determine if the dopaminergic cells of the substantia nigra degenerate by apoptosis in an MPTP mouse model of Parkinson's disease. Methods to identify DNA fragmentation, chromatin condensation, formation of apoptotic bodies and caspase-3 activation accurately detected apoptosis in an X-57 cell system. Preliminary results on brain sections of MPTP treated mice suggest that apoptosis is occurring. This research was performed in the laboratory of Dr. Neil Aronin at UMass Medical School

PLAGL2 Cooperates in Leukemia Development by Upregulating MPL Expression: A Dissertation

Chromosomal alterations involving the RUNXI or CBFB genes are specifically and recurrently associated with human acute myeloid leukemia (AML). One such chromosomal alteration, a pericentric inversion of chromosome 16, is present in the majority of cases of the AML subtype M4Eo. This inversion joins CBFB with the smooth muscle myosin gene MYH11 creating the fusion CBFB-MYH11. Knock-in studies in the mouse have demonstrated that expression of the protein product of the Cbfb-MYH11fusion, Cbfβ-SMMHC, predisposes mice to AML and that chemical mutagenesis both accelerates and increases the penetrance of the disease (Castilla et al., 1999). However, the mechanism of transformation and the associated collaborating genetic events remain to be resolved. As detailed in Chapter 2, we used retroviral insertional mutagenesis (RIM) to identify mutations in Cbfb-MYH11 chimeric mice that contribute to AML. The genetic screen identified 54 independent candidate cooperating genes including 6 common insertion sites: Plag1, Plagl2, Runx2, H2T23, Pstpip2, and Dok1. Focusing on the 2 members of the Plag family of transcription factors, Chapter 3 presents experiments demonstrating that Plag1 and Plagl2 independently cooperate with Cbfβ-SMMHC in vivo to efficiently trigger leukemia with short latency in the mouse. In addition, Plag1 and PLAGL2 increased proliferation and in vitro cell renewal in Cbfβ-SMMHC hematopoietic progenitors. Furthemore, PLAG1 and PLAGL2 expression was increased in 20% of human AML samples suggesting that PLAG1 and PLAGL2 may also contribute to human AML. Interestingly, PLAGL2was preferentially increased in samples with chromosome 16 inversion, t(8;21), and t(15;17). To define the mechanism by which PLAGL2 contributes to leukemogenesis, Chapter 4 presents studies assessing the role of the Mp1 signaling cascade as a Plagl2 downstream pathway in leukemia development. Using microarray analysis we discovered that PLAGL2 induces the expression of Mp1 transcript in primary bone marrow cells that express Cbfβ-SMMHC and that this induction is maintained in leukemogenesis. We have also performed luciferase assays to confirm that the Mp1 proximal promoter can be directly bound and activated by PLAGL2. Furthermore, we demonstrate increased Mp1 expression leads to hypersensitivity to the Mp1 ligand thrombopoietin (TPO) in PLAGL2/Cbfβ-SMMHC leukemic cells. To test the functional relevance in leukemia formation, we performed a bone-marrow transplantation assay and demonstrate that overexpression of Mp1 is indeed sufficient to cooperate with Cbfβ-SMMHC in leukemia induction. This data reveals that PLAGL2 cooperates with Cbfβ-SMMHC at least in part by inducing the expression of the cytokine receptor Mp1. Thus, we have identified the Mp1 signal transduction pathway as a novel target for therapeutic intervention in AML

Somatic genetics empowers the mouse for modeling and interrogating developmental and disease processes.

With recent advances in genomic technologies, candidate human disease genes are being mapped at an accelerated pace. There is a clear need to move forward with genetic tools that can efficiently validate these mutations in vivo. Murine somatic mutagenesis is evolving to fulfill these needs with tools such as somatic transgenesis, humanized rodents, and forward genetics. By combining these resources one is not only able to model disease for in vivo verification, but also to screen for mutations and pathways integral to disease progression and therapeutic intervention. In this review, we briefly outline the current advances in somatic mutagenesis and discuss how these new tools, especially the piggyBac transposon system, can be applied to decipher human biology and disease

Somatic phenotypes like cancer can be modeled and genetically dissected with transposon mutagenesis.

<p>Potential oncogenic pathway to be interrogated with candidate oncogene X and effector Y in red (left). Depiction of <i>PB</i> transposon construct for verifying oncogene X (center). Yellow arrows detail transposon arms. Promoters are depicted by blue pointed boxes. Gene X is indicated by red box and luciferase marker is indicated by green box. To test if effector Y is involved in the oncogenic pathway, an shRNA cassette to knockdown gene Y is represented by the red box (right). The transposons are co-transfected or electroporated with <i>PBase</i> (lower yellow box) to stably integrate the transposon construct into the mouse cells. The green cells in the mouse indicate luciferase positive cells expressing the transposed construct, which are monitored for the tumor formation.</p

Refining the wheelchair prescription process.

Wheelchair prescription is a highly involved process that is unique to each individual. There are many aspects to consider when making a prescription, ranging from environmental needs to the physical needs of the patient. This report, prepared for the Royal Hospital for Neuro-disability, takes an in depth look at the current prescription process. We examine ways in which the RHNd can make the prescription process more standardised and efficient, and explore how an expert system could be beneficial

Screening for phenotypes in humanized mice with patient-derived IPS cells.

<p>IPS cells are first created from a patient. A mutator transposon containing mutagenic elements (red box) and a GFP marker (green box) and an inducible <i>PBase</i> construct (utilizing the Cre-ER/lox or Tet system) is introduced into patient-derived IPS cells. Green cells indicate GFP expression from the stably integrated mutator transposon(s). The cells are then introduced into the mouse tissue by injection (syringe). Next, transposase activity is induced, which mobilizes the mutagenic transposon, resulting in insertional mutation. Finally, the mice are screened for the desired disease or developmental phenotype.</p

The transcription factor PlagL2 activates Mpl transcription and signaling in hematopoietic progenitor and leukemia cells

Cytokine signaling pathways are frequent targets of oncogenic mutations in acute myeloid leukemia (AML), promoting proliferation and survival. We have previously shown that the transcription factor PLAGL2 promotes proliferation and cooperates with the leukemia fusion protein Cbfbeta-SMMHC in AML development. Here, we show that PLAGL2 upregulates expression of the thrombopoietin receptor Mpl, using two consensus sites in its proximal promoter. We also show that Mpl overexpression efficiently cooperates with Cbfbeta-SMMHC in development of leukemia in mice. Finally, we demonstrate that PlagL2-expressing leukemic cells show hyper-activation of Jak2 and downstream STAT5, Akt and Erk1/2 pathways in response to Thpo ligand. These results show that PlagL2 expression activates expression of Mpl in hematopoietic progenitors, and that upregulation of wild-type Mpl provides an oncogenic signal in cooperation with CBFbeta-SMMHC in mice