Rotation Projects for Grad Students
Projects from Fall 2002 and Spring 2003
• Cloning and characterization of retroviral proviral insertion sites in retrovirally-induced mammary tumors and/or myeloid leukemias. Initially this involves PCR reactions and plasmid cloning. Further steps include DNA sequence analysis, bioinformatics, and prediction of the effect of proviral insertion on gene expression. Ultimately, selected genes thought to be involved in cancer will be analyzed in greater detail, including performing functional analysis of the role in cancer.
• Characterization of a new knockout mouse. The gene that has been knocked out is Mds , and represents an alternative start of transcription for the Evi1 gene. This gene is a common site of proviral insertion in myeloid leukemia in mice, and is a site of chromosomal translocation in human myeloid tumors. The gene encodes a zinc finger protein, and when transcription occurs at Mds , a larger isoform is made that has a histone methyltransferase domain at the N terminus. Knockout of the Evi1 locus is homozygous lethat at 9 days of gestation (already published), while knockout of Mds is homozygous viable. However, these mice are small and have a hunched back. This project would involve genotyping of mice, following matings, and further characterization of the Mds homozygous null phenotype. We are also interested in documenting the pattern of expression of the normal Mds transcript during embryogenesis, and how this is altered in the knockout mice. The knockout allele contains the beta galactosidase gene, which can be monitored by histochemical or antibody stains.
• Complementation of the Evi1 knockout by BAC transgenics, and mutagenesis of BACs. We have recently shown that the Evi1 knockout can be complemented with a bacterial artificial chromosome containing the complete Evi1 gene. We are now interested in mutagenizing the BAC to probe the regions of EVI1 that are essential for development. We also wish to create a mouse that makes an EVI1 protein with an affinity tag for purification of EVI1-containing complexes from mouse organs.
• Retrovirus-mediated gene transfer of Evi1 -derived oncogenes into mouse bone marrow to test for leukemogenic potential. We have shown that an AML1/MDS/Evi1 construct, which is the product of the leukemia-associated t(3;21) (in humans), can cause leukemia in mice. Mutations in the functional domains of either AML1 or EVI1 do not destroy the leukemogenic potential. However, it appears that the first set of zinc fingers may be required for leukemogenicity. This project involves the harvesting oof mouse bone marrow, infection with retrovirus, and transplantation back into irradiated recipients. Further analysis of the resultant tumors is also possible. In the course of this project, you will gain an appreciation for the differentiation of hematopoietic cells and the pathogenesis of leukemia.
• Identification of EVI1 target genes. We have developed a number of cell lines in which the EVI1 gene can be induced. We have also been able to inhibit Evi1 expression in mouse leukemias that have proviral integrations at Evi1 using small inhibitory RNAs.Messenger RNAs from these cells can be applied to cDNA or oligo microarrays to identify those genes that are responsive to EVI1.
Analysis of gene expression in mouse leukemias. We have banked sets of leukemias from mice that are due to different oncogenic events. We are interested in analyzing these with cDNA and oligo microarrays to see what patterns exist. This will involve extensive use of cDNA microarrays as well as bioinformatics.