Cell engineering method using fluorogenic oligonucleotide signaling probes and flow cytometry.

OBJECTIVE: Chromovert® Technology is presented as a new cell engineering technology to detect and purify living cells based on gene expression. METHODS: The technology utilizes fluorogenic oligonucleotide signaling probes and flow cytometry to detect and isolate individual living cells expressing one or more transfected or endogenously-expressed genes. RESULTS: Results for production of cell lines expressing a diversity of ion channel and membrane proteins are presented, including heteromultimeric epithelial sodium channel (αßγ-ENaC), sodium voltage-gated ion channel 1.7 (NaV1.7-αß1ß2), four unique γ-aminobutyric acid A (GABAA) receptor ion channel subunit combinations α1ß3γ2s, α2ß3γ2s, α3ß3γ2s and α5ß3γ2s, cystic fibrosis conductance regulator (CFTR), CFTR-Δ508 and two G-protein coupled receptors (GPCRs) without reliance on leader sequences and/or chaperones. In addition, three novel plasmid-encoded sequences used to introduce 3' untranslated RNA sequence tags in mRNA expression products and differentially-detectable fluorogenic probes directed to each are described. The tags and corresponding fluorogenic signaling probes streamline the process by enabling the multiplexed detection and isolation of cells expressing one or more genes without the need for gene-specific probes. CONCLUSIONS: Chromovert technology is provided as a research tool for use to enrich and isolate cells engineered to express one or more desired genes.

Nuclear localization and dynamic properties of the Marek's disease virus oncogene products Meq and Meq/vIL8.

Marek's disease virus (MDV) is an avian herpesvirus that causes T-cell lymphomas and immune suppression in susceptible chickens. At least one gene product, MDV Eco Q-encoded protein (Meq), is essential for the oncogenicity of MDV. Alternative splicing permits the meq gene to give rise to two major transcripts encoding proteins designated Meq and Meq/vIL8. Meq is a basic leucine zipper protein capable of modulating transcription. The Meq/vIL8 protein retains a modified leucine zipper, along with the mature receptor-binding portion of vIL8, but lacks the domain of Meq responsible for transcriptional modulation. In this report, we describe studies using fusions between either Meq or Meq/vIL8 and fluorescent proteins to characterize the distribution and properties of these products in chicken embryo fibroblasts (CEFs). Meq and Meq/vIL8 both localized to the nucleoplasm, nucleoli, and Cajal bodies of transfected cells. Similar distributions were found for fluorescent fusion proteins and native Meq or Meq/vIL8. Fluorescence recovery after photobleaching and photoactivatable green fluorescent protein revealed that Meq exhibited mobility properties similar to those of other transcription factors, while Meq/vIL8 was far less mobile. In addition, fluorescence resonance energy transfer studies indicated the formation of Meq/vIL8 homodimers in CEFs. Time lapse studies revealed the coordinated elimination of a portion of Meq and Meq/vIL8 from the nucleus. Our data provide new insight regarding the dynamic cellular properties of two forms of a herpesvirus-encoded oncoprotein and suggest that these forms may have fundamentally different functions in MDV-infected cells.

Herpesvirus of turkeys: microarray analysis of host gene responses to infection.

Herpesvirus of turkeys (HVT) provides an economically important live vaccine for prevention of Marek's disease (MD) of chickens. MD, characterized by both immunosuppression and T-cell lymphoma, is caused by another herpesvirus termed Marek's disease virus (MDV). Microarrays were used to investigate the response of chicken embryonic fibroblasts (CEF) to infection with HVT. Genes responding to HVT infection include several induced by interferon along with others modulating signal transduction, transcription, scaffolding proteins, and the cytoskeleton. Results are compared with earlier studies examining the responses of CEF cells to infection with MDV.