Progress 07/01/00 to 12/31/04
Outputs
Gene tagging is a strategy used to randomly capture functional gene units using a vector linked to a reporter and selection system. Gene trap DNA vectors were re-designed to incorporate a splice acceptor (SA) sequence at the junction to the LacZ reporter read-out. LacZ would be expressed if an active gene promoter were trapped. A vector containing the same structure but with LacZ replaced by GFP was also designed. The vector pieces were subcloned with the neomycin cassette inserted downstream. The junctions were sequenced. Electroporation of the "gene trap" DNA vector into ES cells for expression was done to see if the re-design had produced vigorous reporter expression. After selection in G418 medium to identify the clones that had captured the "gene trap", they were picked into 24 well plates for expansion. An aliquot of the expanded cells was tested for LacZ expression. There were no colonies expressing LacZ. These results suggest that the 1) the vector design is
still not optimal, 2) this system simply does not capture strong expression from a promoter or 3) the trapped clones are dying somewhere in the culture process. Since these studies were undertaken, technology incorporating ENU mutagenesis with sperm and breeding through the F2 generation has been gaining ground as a technique to link genes with function .
Impacts
Genes with functional can be trapped and "knocked out" randomly without prior knowledge of gene sequence or identity. New and novel genes may be identified in the course of these experiments.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
Gene tagging is a strategy used to randomly capture functional gene units using a vector with a splice acceptor (SA)/donor (SD) sequence linked to a reporter and selection. DNA vectors were re-engineered with a SA/SD linked to an LacZ readout that would be activated if an active gene promoter were trapped. This vector was electroporated into J1 ES cells and selected in G418 medium. An estimated 300 ES clones were obtained after selection. These were picked and expanded 1) for viable freeze and 2) for LacZ expression. Only two clones gave week lacZ expression and these were not sufficiently beyond background to be used to produce transgenic mice for analysis. This system can be optimized by redesigning the tarp vector to include EGFP expression in place of the LacZ. Then clones that are growing under G418 expersion can be examined directly for "trapping". Only those clones with a clear trap phenotype need be saved and manipulated for further transgenic analysis.
Impacts
Genes with functional can be trapped and "knocked out" randomly without prior knowledge of gene seqeunce or identity. New and novel genes may be identified.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
The project's goal is to develop a binomial gene tagging method to capture location and putative function of unknown genes in vivo. Reporter ES cell lines (random inserts of LacZ under the control of captured endogenous promoter/enhancer regions) are developed and used to produce murine chimera. The LacZ readout locates the tissue and expression pattern of the tagged gene. A new gene tagging construct with a GFP/Neo cassette design (IRES preceding Neo) was tested by electroporation into J1 ES cells. Selection in G418 was toxic to the ES cells indicating that the IRES does not support transcription of the Neo cassette. Additional electroporations with this construct and a dose response curve of G418 are being tested.
Impacts
Gene tagging to identify the function of undescribed genes relies on the the capture of active promoter/enhancer combinations at selected time points in development. It is an alternative approach to the large ethyl nitrosourea (ENU) mutageneis screens and captures a few individual genes. It also has the potential to uncover recessive /knockout phentypes as well as dominant mutations.
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
The project's aim is to develop a binomial gene tagging method and construct(s) to capture the location and putative function of uncharacterized genes in vivo. A novel gene tagging strategy combines a reporter mouse derived from inserting a GFP or b-galactosidase construct randomly into ES cells. These reporter ES cells are used to produce murine chimera . Germline transmission and breeding to homozygosity for the inserted genotype establish the reporter mouse line. The reporter construct (Lox2Neo/b-Gal) was subcloned and electroporated into the J1 ES cell line. ES cell clones containing the construct were selected in culture using G418 (n=123 NeoR clones). The functionality of each clone was tested in vitro for its endpoint---expression of b-galactosidase--- by a second electroporation of the reporter activating DNA construct pBS185 (hCMV-Cre) . The results from this experiment show that the reporter construct is inactive in its current design. A new subcloning
strategy is under consideration. In this binomial system, a gene tagging promotorless construct (Splice donor/Splice acceptor-Cre recombinase-pA) is required to complete the system. This construct is being assembled from available DNA fragments and will be microinjected into pronuclei of murine embryos.
Impacts
Murine genomic studies are using large chemical mutagenesis screens to capture bioinformation on genes of unknown function. These studies primarily capture dominant-negative mutations using a phenotype screen. The binomial system under development in this project has the potential to uncover recessive/knockout phenotypes from gene-tagged mutants as well as dominant mutations. It should also provide some tissue/organ localization information from the inclusion of the b-galactosidase gene in the reporter mouse line. In addition, screening early embryos for reporter expression may uncover genes active in early development before the "trap " produces an embryonic lethal.
Publications
- No publications reported this period
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Progress 01/01/00 to 12/31/00
Outputs
The projects aim is to develop a binomial gene tagging method and construct(s) to capture the location and putative function of uncharacterized genes in vivo. The technology employs a novel gene tagging strategy that will be combined with development of transgenic mouse models. To date, the gene-tagging construct fragments are subcloned into a single plasmid (lox2Neo/beta-Gal). This gene-tagging construct was constructed by inserting gene cassettes into a single plasmid containing a large MCS. This design permits removal and insertion of other reporter cassettes as needed. For example, the beta-galactosidase cassette can be switched with an enhanced green fluorescent protein (EGFP) reporter cassette, if necessary. The lox2Neo cassette is inserted between the CMV promoter and the beta-galactosidase reporter and is used as a STOP signal. In this binomial system, the galactosidase reporters expression is activated only after the excision of the lox2Neo cassette by a
promoter trapped Cre construct. Currently, the beta-galactosidase reporter construct is being tested in an ES cell culture system for activation by CMV-Cre. The ES cell culture system is also being used to examine whether there is any "leaky" expression when the Lox2Neo cassette is used as a STOP. The gene-tagging construct also is being subcloned. It will be used as a promoter trap construct. It will also be tested for activity in the ES cell culture system prior to use in transgenic mice.
Impacts
Murine genomic studies are using large chemical mutagenesis screens to capture bioinformation on uncharacterized genes. These studies primarily capture dominant-negative mutations in the phenotype screen. The binomial system under development in this project has the potential to uncover recessive/knockout phenotypes from gene-tagged mutants as well as dominant mutations. It should also provide some tissue localization information from the inclusion of the beta-galactosidase reporter
Publications
- No publications reported this period
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