| Laboratory of Molecular Gerontology |
Intramural Program Investigators ^ Home ^ |
| Michael Seidman, Ph.D., Senior Investigator Chief, Section on Gene Targeting |
 Dr. Michael Seidman received his Ph.D. in biochemistry from the University of California, Berkeley, in 1975. He did postdoctoral work at the NIH and at Princeton University. He worked at the NIH and in the biotechnology industry before assuming his present position in the Laboratory of Molecular Genetics, NIA.Research Interests: Cellular Response to DNA Damage: We are interested in the response of cells to targeted DNA damage and the application of site specific targeting for modulating genomic sequences. Gene Targeting: Current approaches for manipulating genomic sequences rely on homologous recombination. In these procedures relatively lengthy DNA fragments are introduced into cells and via an enzymologically driven process engage in a search for homologous sequences in the chromosome. After a recombinational intermediate forms, the process is completed in a series of additional enzymatic steps. The procedure is inefficient and time consuming. Given the marked increase in sequence data from the genome project there is a clear utility in having a more efficient and less cumbersome process. |
| We are developing oligonucleotides, that can form a three-stranded DNA structure called a triple helix. The third strand lies in the major groove of an intact double helix and is stabilized by hydrogen bonds between the bases in the third strand and the purine bases in the duplex. These structures are quite stable and very stringent with respect to sequence specificity. The oligonucleotides can be linked to DNA reactive compounds and site-specific modification of DNA with these oligo-reagent conjugates has been demonstrated by many groups. Although these structures have been studied for many years, there have been relatively few accounts of biological applications. |
| Recently we and our colleagues constructed an oligonucleotide linked to psoralen (a photoactive DNA crosslinker), which was designed to form a triplex with a sequence in the well-known cellular housekeeping gene HPRT (hypoxanthine guanine phosphoribosyl transferase). This gene encodes an enzyme engaged in purine salvage. There is a simple selection procedure for cells, which lack the enzyme, consequently, the gene has become the most commonly used mutation marker gene in mammalian cells. The oligo was introduced in cells in culture and after photoactivation the cells were processed according to a standard mutation selection protocol. Mutations were found at the target site, and sequence analysis demonstrated that the majority were small deletions. This was the first evidence that chromosomal targets are accessible to triplex forming oligonucleotide reagents. |
| In more recent work we have examined the influence of novel sugar modifications on the activity of triplex forming oligonucleotides. We have identified the nature and distribution of these derivatives in oligonucleotides that support robust activity in gene knockout assays. We are now using these new reagents in additional gene knockout studies, as probes of cellular chromatin structure, and for studying the metabolism of targeted DNA damage. |
| This approach can now be used to deliver additional DNA reactive compounds to specific genomic locations and we are in the process of developing those reagents. We are also looking at the influence of DNA repair deficiencies on the targeted mutation frequencies. This will tell us which DNA repair activities are active in repair of the directed lesions, and lead to the development of strategies designed to inhibit these functions during the time of oligo introduction. Eventually this approach will be used to modulate genomic sequences with targeted gene knockout as a specific application. |
| Contact Information: Laboratory of Molecular Gerontology Biomedical Research Center, room 05B133 251 Bayview Boulevard, Suite 100 Baltimore, MD 21224-6825 Phone 410-558-8565 Fax 410-558-8157 E mail seidmanm@grc.nia.nih.gov For more information about the Laboratory: http://www.grc.nia.nih.gov/branches/lmg/lmg.htm |
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