X-linked severe combined immunodeficiency (X-SCID) is caused by a mutation in the interleukin 2 receptor gamma (IL2Rγ) gene, which is required for lymphocyte development. Affected patients lack functional lymphocytes and die unless cured through bone marrow transplant or gene therapy. However, gene therapy treatment was stopped due to risk of leukemia resulting from insertional mutagenesis, and this prompted the pursuit of alternate treatments, such as gene repair. Gene repair requires the use of two essential components, a homing endonuclease and a repair template, that harness homology directed repair mechanism, which repairs double stranded DNA breaks in cells. Homing endonucleases are enzymes used to target and cut DNA near a mutation in the genome, while a repair template is the DNA sequence used to correct the mutation. My project involved performing experiments to optimize specific components of gene repair using the Sce-SCID mouse model that contained a recognition sequence for the homing endonuclease, I-SceI proximal to the IL2Rγ mutation. I used adeno-associated viral vectors (AAV) to deliver I-SceI and a repair template to hematopoietic stem cells (HSC) isolated from Sce-SCID mice. I tested the hypothesis that delivery of AAV encoding I-SceI homing endonuclease to Sce-SCID HSC would result in recognition and cutting of the Sce locus. Cutting of the Sce locus is essential to gene repair because it initiates the repair of DNA double stranded breaks through homology directed repair with the repair template. Previous experiments in the Rawlings lab demonstrated cutting of the Sce locus with stable I-SceI expression, but my experiments involved transient AAV delivery. Transient delivery is critical for the safety profile of gene repair. Once I-SceI cutting was established with transient delivery, I performed experiments that invovled both I-SceI and repair template in an attempt to correct the SCID phenotype of the Sce-SCID mouse.

Diatoms are unicellular photosynthetic algae, or phytoplankton, and account for approximately 20% of global primary production. The diatom Pseudo-nitzschia can produce a neurotoxin called domoic acid (DA) that builds up in the tissues of shellfish when this diatom blooms. DA poisoning causes life-threatening conditions in mammals and humans when these shellfish are ingested. Parameters such as shellfish exposure length and bloom toxicity can be found by understanding what regulates diatom communities. One mechanism of bloom regulation that we know little about is that of viral infection, despite viruses being the most abundant predator in the ocean. My lab is isolating and characterizing viruses to develop a model system. After a toxic Pseudo-nitzschia bloom at Sunset Beach, Oregon was sampled in 2009, the first Pseudo-nitzschia infecting virus was isolated by infecting the host P. multiseries Clnn-16. This virus was named PmDNAV and was characterized by its burst size, latent period, host range, morphology, and nucleic acid content. I hypothesize that there are many different viruses that can infect diatoms of the genus Pseudo-nitzschia in addition to the PmDNAV. My project focuses on isolating and characterizing more viruses, in order to compare them to the PmDNAV. I have samples from 4 different blooms and 43 different hosts in culture to test these lysates on. I found that the host Clnn-16 dies when inoculated with lysates from the Sunset Beach sampling. I repeated this experiment to prove that my lysates could infect a new healthy host. I am working to prove that this death is due to a virus by looking for small pinpricks of light on a slide after staining the nucleic acids of the putative viruses. After that, I will make the virus clonal and work to characterize this virus, later repeating this process with as many viruses as possible.

Synechococcus is a unicellular cyanobacterium about 1µm in diameter. It is an abundant photosynthetic microorganism and a great contributor to the primary productivity of the Earth’s oceans. Synechococcus communities are composed of different phylogenetic clades that are believed to represent physiologically and genetically diverse populations. Previous data has shown two phylogenetically distinct populations of Synechococcus, clade I and clade IV, to be dominant in Puget Sound, with fluctuations in these populations during different seasons and regions. For my research, I will attempt to identify a clade IV strain. In an attempt to enhance conditions where clade IV will be most competitive, I have varied the culturing parameters such as using multiple media types. Filtered whole water samples containing mixed assemblages of Synechococcus were collected from Puget Sound and were initially cultured in natural Puget Sound seawater. These cultures were poured onto agar plates with sterilized natural Puget Sound water as well as plates with artificial seawater with different concentrations of AMP1 nutrients. The 16S rDNA of the isolates identified as clade IV will be sequenced by the use of PCR using primer sets that target specifically Synechococcus clade I or clade IV. The success, or failure, of amplification will indicate whether the Synechococcus DNA template was Synechococcus clade I or Synechococcus clade IV. Any Synechococcus DNA that results in a successful amplification using the Synechococcus clade IV primer set, I will send off to be sequenced. We have nutrient and physical measurements for these samples to help explain the environmental parameters that control distribution of these populations. With this information and ITS sequences I can compare Synechococcus clade IV populations within Puget Sound, and compare these with coastal populations that have been previously isolated. We seek to understand how these populations fluctuate seasonally and throughout different regions of the Sound.