Dr. Sood's research is aimed towards developing resources and adopting new technologies to facilitate the functional analysis of genes involved in human genetic diseases using the zebrafish as a model organism. Her goal is to adopt new technical developments in the field of zebrafish research so that all NHGRI investigators can capitalize on these cutting-edge methodologies.
Animal models with gain or loss of function mutations in genes play an important role in functional genomic studies, developing models for understanding the pathophysiology of diseases, and novel therapeutic approaches. To achieve this goal using the zebrafish as a model system, Dr. Sood utilizes two complementary approaches: random mutagenesis using N-ethyl-N-nitrosourea (ENU) induced point mutations termed TILLING (for "targeting induced local lesions in genomes") and targeted mutagenesis using zinc-finger and tal-effector nucleases (ZFNs and TALENs). Most of the mutations identified by TILLING approach are missense in nature. On the other hand, mutations induced by ZFNs and TALENs are small insertions or deletions leading to frameshift with premature truncation of the protein. Thus the choice of mutagenesis method depends on the nature of desired mutations in the gene of interest. In both approaches, once the desirable mutant alleles are identified, in depth phenotype analysis is performed by morphological, histological and biochemical assays.
For TILLING, Dr. Sood's laboratory has generated DNA and cryopreserved testes from ~3500 zebrafish males heterozygous for ENU-induced random point mutations throughout their genomes. Reverse genetic approaches involving polymerase chain reaction (PCR) and sequencing of exons coding for functional domains are used to identify mutations in genes of interest followed by generation of new fish lines using corresponding testes sample. To date, Dr. Sood has generated over two dozen lines with missense and truncation mutations in genes involved in diverse cellular and developmental processes such as DNA repair, hematopoiesis, gastrulation and cancer.
For targeted mutagenesis, Dr. Sood's group microinjects mRNA-encoding ZFNs or TALENs specifically targeting the gene of interest into one-cell stage zebrafish embryos. The injected embryos, or "founders," are grown to adulthood, and screened for germline transmission of mutations induced by the ZFNs or TALENs. Progeny from germline transmitting founders is grown to adulthood to identify the heterozygous fish. Dr. Sood's laboratory has developed high throughput protocols for efficient generation of mutations using ZFNs and TALENs. To date, Dr. Sood has generated multiple knockout mutant alleles for over a dozen genes studied by her own laboratory and other researchers at NHGRI.
Dr. Sood has a long-standing interest in understanding the process of cancer development and progression. In parallel with the technical development work being conducted in the Zebrafish Core, Dr. Sood has collaborated with Dr. Paul Liu to characterize the phenotypes of zebrafish mutations in genes involved in hematopoiesis; mutations in several genes that control hematopoiesis cause leukemias and lymphomas.
Through the study of novel zebrafish mutations in gata1 and runx1, two of the major regulators of primitive and definitive hematopoiesis, respectively, Drs. Sood and Liu have demonstrated differential requirements of these genes during different waves of hematopoiesis. These mutants provide tools for clarifying the mechanism of hematopoiesis and developing new therapeutics by high-throughput chemical screening.
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Last Updated: May 18, 2014