I am working with Bacterial Artificial Chromosomes (BACs) which were screened using primers designed from known regions of the cDNA. BACs verified to contain both the 3' and 5' regions have been isolated. Human genomic DNA was digested and ligated into Bluescript plasmid. Plasmids were isolated from the transformed cells and PCR was used to confirm the presence of the gene. PCR was used to amplify the gene and the surrounding genomic DNA, which was sequenced.

Adenosine deaminase deficient severe combined immunodeficiency (ADA-SCID) was the first disease investigated for gene therapy. ADA-SCID is an autosomal recessive disorder which results in a lack of ADA enzyme. In 1990, a clinical trial was started using retroviral mediated transfer of the ADA gene into the T cells of two children, resulting in improved cellular and humoral responses. Seven years after the last gene therapy treatment, the integrated vector and ADA expression persisted. In 1998, T cells were obtained and single cell clones were established, using limiting dilution. Because the patients were infused multiple times with ADA retroviral vector LASN transduced autologus T cells, we wanted to determine how many copies of the vector had integrated into the T cells. For this purpose, I am analyzing the clones using PCR and the ABI GeneScan/ Genotyper programs. My first step is to determine the linear range of the PCR reaction for the vector and the internal control amplicons. Then I will perform PCR reactions at the optimal cycle number. Using peak height values from GeneScan, copy number will be determined using the ratio of the integrated vector: internal control.

I am currently constructing a database of all known genes that are involved in the development, function and regulation of bone and skeletal tissues. This project is part of a continuing effort and will be used by the Skeletal Genome Anatomy Project (SGAP) to determine which genes have already been identified and sequenced as well as those which are novel.

This summer I have been working with the OS4 novel protein. OS4 is a signifigant gene to study because although it has no clear identity, such a broad spectrum of expression suggests that OS4 has an essential role. Through my project I have expressed two OS4 constructs: OS4-thiroedoxin fusion protein and OS4-GST fusion protein. Antibodies were tested for localization of protein in order to study protein interactions. The techniques utilized were western blotting and protein purification. Some samples were run on agarose mini-gels to test for accuracy of the constructs. Ultimately, the purpose of the project is to determine the function of the OS4 protein.

The research performed was part of the Skeletal Genome Anatomy Project. The goal of this project is to characterize novel genes espressed in human trabecular bone cells using novel EST's (Expressed Sequence Tags). Various molecular techniques are used to assist in defining size, location, the full-length DNA sequence, genomic structure and organization, and to provide insight to the function of the protein of the novel gene.

X-Linked Severe Combined Immunodeficiency (XSCID) is an inherited disorder that severely hinders an individual's ability to combat infectious disease. The primary characteristics of XSCID are a lack of circulating T-lymphocytes in the peripheral blood, failure of T-cells to mature in the thymus, and a virtual lack of cellular and humoral immunity. While B-lymphocytes are generally present in normal numbers, they are abnormal in their function. XSCID is caused by mutations in the gene that encodes for the gamma-chain of the Interleukin-2 (IL-2) receptor, which is presented extracellularly of leukocytes. The gamma-chain is also a component of the receptors for many other cytokines, including IL-4, IL-7, IL-9, and IL-15, thus giving it the name of the "common gamma-chain" (gc). IL-7 is dependent upon gc for signal transduction, and is necessary for T-cell maturation and development in the murine thymic organ culture system. It is not yet known whether the latter holds true for human thymopoiesis, although it would explain the observed lack of peripheral T-cell in XSCID patients. Currently, the primary treatment for XSCID is allegenic bone marrow transplantation. However, due to lack of compatible marrow donors, as well as the risk involved in the procedure, this treatment is not available to everyone. As an alternative to bone marrow transplantation, gene therapy is being developed as a potential treatment for XSCID. Initial studies involved the use of retroviruses, which scientists used to deliver normal gc to XSCID patients cells. There is a problem in using a retrovirus to treat diseases such as XSCID; retroviruses are not able to transduce non-dividing cells. However, there is a family of viruses that can efficiently transduce dividing as well as non-dividing cells- the lentiviruses, which are the family of viruses that include HIV-1. Our objective was to build a lentiviral-based vector containing gc to correct XSCID B-cells by inserting into the cellular DNA a copy of the wild-type gc gene.

My research involved identifying the cause of a particular case of Glycogen Storage Disease Type VI (GSD6) in the child of unrelated parents. Glycogen storage diseases, in general, are caused by defects in the liberation of glucose from glycogen. GSD6 or Hers Disease is a very rare disorder that has shown to be inherited in a genetically recessive manner. The manifestation of GSD6 is usually mild, affecting infants in their first few years of life. Common symptoms include hepatomegaly, growth retardation, and irritability. The disease usually takes a benign course, with remission of symptoms as the affected children grow up. While the disease is usually mild, the diagnosis is at times extremely intrusive. Identification of the genetic cause of the disease could allow for a simple, unobtrusive diagnostic test. In order to determine the genetic cause of the disease, the liver glycogen phosphorylase (PYGL) gene was examined for mutations. PYGL was determined to be a good candidate gene because of its reduced levels in the liver in GSD6, and its involvement in the catalytic degradation of glycogen to glucose-1-phosphate. This gene was sequenced in four subjects: an affected child, the parents of the affected child, and an unrelated wild type. The sequences of the four subjects are currently being compared in order to identify if any mutations exist that could cause GSD6. It is expected that the affected child will be a compound heterozygote, having a different mutation on the two alleles, and that the parents will each have one of the mutations.

I am studying the MDR1 gene (multi-drug resistance gene) on chromosome 7. The protein encoded by this gene helps to form the blood-brain barrier, which prevents certain substances from entering the brain. Some people develop central nervous system toxicity with drugs known to be transported by the MDR1 protein. We thus hypothesized that genetic differences in MDR1 may account for this variable toxicity. My project involved searching for single-nucleotide polymorphisms (SNPs) within the MDR1 gene, with the long-term aim of studying the sequence of MDR1 in numerous people, including those that have developed drug-induced central nervous system toxicity. This work involved developing and optimizing PCR assays across the entire MDR1 coding region and sequencing the resulting products.

Rlk is a tyrosine kinase expressed in T-cells and is related to Btk, the kinase mutated in X-linked agammaglobulinemia. We have shown that Rlk is phosphorylated and activated by the Src family kinase Fyn. Coexpression of Rlk and Fyn leads to increased Rlk tyrosine phosphorylation and kinase activity. To determine requirements for Rlk activation, we mutated the SH2, SH3, and Proline Rich regions of Rlk. We found that the Rlk SH2 mutant appears less tyrosine phosphorylated when expressed with Fyn. To determine if the SH2 mutant prevents Fyn phosphorylation or decreases Rlk kinase activity we introduced the SH2, SH3, and Proline mutants into kinase dead Rlk. These experiments may provide insight into the activation and function of Rlk in T-cells.

My project involved helping to sequence a bacterial artificial chromosome (BAC) containing human DNA, similar to that being performed for sequencing the human genome as part of the Human Genome Project. This process starts with taking BAC DNA and shearing it into small fragments, and inserting the resulting fragments into plasmid or M13 vectors. Following transformation into E. coli, resulting subclones are selected, and DNA from each subclone is purified and subjected to fluorescent-based DNA sequencing. The latter involves synthesizing new DNA in the presence of fluorescently labeled dideoxy nucleotides, with each of the four dideoxy bases (G, A, T, C) containing a differently colored fluorescent dye that emits light at a different wavelength when excited with a laser. The fluorescently labeled DNA is analyzed on an acrylamide gel, such that smaller-sized DNA strands travel faster than the larger one. The gel effectively resolves DNA fragments that differ by only one base in size. A laser detects the DNA as it moves down the gel, allowing the color of the dideoxy nucleotide (and therefore the base at that position) to be determined. By analyzing hundreds of subclones from each BAC, it is possible to eventually assemble the sequence of the starting BAC by detecting overlaps among the different subclones.

Prostate cancer is the second most common cause of cancer death in males in the United States. Despite much research on advanced prostate cancer, there is no known etiology and no available cure for the disease. The aim of this study is to apply cDNA microarray technology to identify gene expression changes that might account for the androgen independent growth of prostate cancer cells. One of the challenges of using cDNA microarray technology is performing experiments in such a way that multiple tumors can be compared rather than simply comparing paired samples. In this project, we developed a system whereby we use a standard mRNA pool against which we compare the mRNA profile of several tumors. Large quantities of LNCap cells, an androgen sensitive tumor cell line, were propagated. Poly A mRNA was isolated from these cells and pooled for use as a standard in future microarray experiments. This mRNA will now be used against various human prostate cancer cell mRNA to identify genes which are differentially expressed and may result in androgen independent growth.

I worked in the Bioinformatics Group of the NIH Intramural Sequencing Center (NISC). The primary focus of NISC is to provide NIH investigators access to production-scale DNA Sequencing. The Bioinformatics Group focuses on the assimilation and analysis of the generated DNA sequence. My summer project involved designing a Java-based program, called ChromatogramViewer, that will allow NIH investigators the ability to view their NISC-generated sequence traces over the web.

I am working in the Bioethics and Social Populations Research Program on a project entitled "The Social Construction of Benefit in Gene Transfer Research." The aim of the study is to learn how subjects who participate in gene transfer research benefit from being part of the protocol and how these benefits are understood as well as discussed by principal investigators, study coordinators, IRBs (Institutional Review Boards), patients, and consent forms. This summer, consent forms and protocol summaries for all gene therapy protocols submitted to the Recombinant DNA Advisory Committee from 1990 to the present will be collected and reviewed. The data analyzed will provide important information on how benefits are conveyed to subjects participating in gene therapy research.

I am currently working on a project involving gene therapy for patients with sickle cell disease. Sickle cell disease is caused by mutations in one of the hemoglobin genes leading to polymerization of the hemoglobin in red blood cells, causing the classic sickle shape for which the disease is named. Expression of the gamma-globin gene should prevent the sickling of the red cells that occurs in sickle cell patients. Retrovirus vectors have been used to insert the gamma-gene into mouse blood forming cells. Analysis of these mice has demonstrated gamma-globin expression at levels thought to be almost high enough to cure sickle cell disease. Future challenges include getting higher levels of gamma gene expression and to reproduce this result in human blood forming cells.

Mutations in different domains of FGFR3 are associated with a number of skeletal disorders. Among them are the substitutions of glutamic acid, methionine, and asparagine at Lys650 that are found in patients with thanatophoric dysplasia type II (TDII), severe achondroplasia with developmental delay and acanthosis nigricans (SADDAN), and hypochondroplasia (HCH), respectively. In order to characterize the effects of these three mutations upon the signaling pathway through which FGFR3 regulates cell growth, the Lys644Asp, Lys644Met and Lys644Glu mutants of mouse FGFR3 (corresponding to human Lys650) were transiently expressed in human embryonic kidney 293 cells. In vitro autophosphorylation experiments showed that FGFR3 with Lys644Met mutation was activated twice as high as the Lys644Glu mutant, which did not correspond to the severity of human phenotypes. Abnormally high overall phosphorylation was observed in cells expressing Lys644Met mutant, suggesting the involvement of an abnormal signaling pathway. Indeed, abnormal phosphorylation of STAT family members and Tyk2 was observed in cells expressing FGFR3 with Lys644Met mutation.

I am working on the role of the fusion gene CBFB-MYH11 in leukemia development. Recently, a new line of Cbfb-MYH11 conditional knock-in mice were generated in our laboratory using the Cre-Lox system. These mice do not express the fusion oncogene during embryogenesis and its expression can be turned on in adult mouse by the induction of Cre expression. Exon 5 and 6 flanked by Lox sequences were inserted in intron 4. Thus, the presence of this insertion, prevented Cbfb-MYH11 expression, so pups heterozygous for the gene were born and reach adulthood. We then bred these mice with transgenic mice carrying inducible Cre enzyme (MX1 and fes-Cre). The presence of Cre enzyme deletes the region betweeen Lox sites, and Cbfb-MYH11 is expressed. We aim to test for the relevance of Cbfb-MYH11 expression in adult hematopoietic progenitors in the development of leukemia. We are also crossing these conditional knock-in mice with p53 knock-out mice to study the effect of p53 in this type of leukemia.

I am assisting in the construction of relevant, accurate patient information on medical records and submitting this data into a patient database. I will also be attending clinics and observing genetics cases. I have also been invited to make observations in a molecular lab as well as a cytogenetics lab.