Large-scale DNA Barcode Library Generation for Biomolecule Identification in High-throughput Screens.

High-throughput screens allow for the identification of specific biomolecules with characteristics of interest. In barcoded screens, DNA barcodes are linked to target biomolecules in a manner allowing for the target molecules making up a library to be identified by sequencing the DNA barcodes using Next Generation Sequencing. To be useful in experimental settings, the DNA barcodes in a library must satisfy certain constraints related to GC content, homopolymer length, Hamming distance, and blacklisted subsequences. Here we report a novel framework to quickly generate large-scale libraries of DNA barcodes for use in high-throughput screens. We show that our framework dramatically reduces the computation time required to generate large-scale DNA barcode libraries, compared with a naїve approach to DNA barcode library generation. As a proof of concept, we demonstrate that our framework is able to generate a library consisting of one million DNA barcodes for use in a fragment antibody phage display screening experiment. We also report generating a general purpose one billion DNA barcode library, the largest such library yet reported in literature. Our results demonstrate the value of our novel large-scale DNA barcode library generation framework for use in high-throughput screening applications.

Use of cell type-specific transcriptome to identify genes specifically involved in Müller glia differentiation during retinal development.

Retinal progenitor cells alter their properties over the course of development, and sequentially produce different sub-populations of retinal cells. We had previously found that early and late retinal progenitor cell populations can be distinguished by their surface antigens, SSEA-1 and c-kit, respectively. Using DNA microarray analysis, we examined the transcriptomes of SSEA-1 positive cells at E14, and c-kit positive, and c-kit negative cells at P1. By comparing data, we identified genes specifically expressed in c-kit positive late retinal progenitor cells. The previous literature suggests that most of the c-kit positive cell-specific genes are related to glia differentiation in brain or are expressed in Müller glia. Since Notch signaling promotes Müller glia differentiation in retina, we examined the effects of gain- and loss-of-Notch signaling on expression of these genes and found that all the genes were positively affected by Notch signaling. Finally, we screened the genes for their function in retinal development by shRNA-based suppression in retinal explants. In about half the genes, Müller glia differentiation was perturbed when their expression was suppressed. Taken together, these results show that at P1, c-kit positive retinal progenitor cells, which include Müller glia precursor cells, are enriched for genes related to glial differentiation. We propose analysis of purified subsets of retinal cells as a powerful tool to elucidate the molecular basis of retinal development.

Identification of FoxR2 as an oncogene in medulloblastoma.

Medulloblastoma is the most common pediatric brain tumor, and in ∼25% of cases, it is driven by aberrant activation of the Sonic Hedgehog (SHH) pathway in granule neuron precursor (GNP) cells. In this study, we identified novel medulloblastoma driver genes through a transposon mutagenesis screen in the developing brain of wild-type and Trp53 mutant mice. Twenty-six candidates were identified along with established driver genes such as Gli1 and Crebbp. The transcription factor FoxR2, the most frequent gene identified in the screen, is overexpressed in a small subset of human medulloblastoma of the SHH subtype. Tgif2 and Alx4, 2 new putative oncogenes identified in the screen, are strongly expressed in the SHH subtype of human medulloblastoma. Mutations in these two genes were mutually exclusive with mutations in Gli1 and tended to cooccur, consistent with involvement in the SHH pathway. Notably, Foxr2, Tgif2, and Alx4 activated Gli-binding sites in cooperation with Gli1, strengthening evidence that they function in SHH signaling. In support of an oncogenic function, Foxr2 overexpression transformed NIH3T3 cells and promoted proliferation of GNPs, the latter of which was also observed for Tgif2 and Alx4. These findings offer forward genetic and functional evidence associating Foxr2, Tgif2, and Alx4 with SHH subtype medulloblastoma.