Quantitative RT-PCR to evaluate in vivo expression of multiple transgenes using a common intron.

An assay measuring RNA expression levels of a gene-encoded therapeutic must distinguish between endogenous mRNA and mRNA transcribed from the transgene. Specificity for the delivered transgene is especially critical when the treatment involves genes that are expressed in the target tissue. To facilitate uniform detection of transgene RNA without interference from endogenous mRNA, we have engineered expression vectors that include a 5' untranslated region (5' UTR) containing a synthetic intron (PGL3). The synthetic intron splice junction was the target sequence for a quantitative reverse transcription (RT)-PCR assay utilizing Taq-Man technology. In this study, we demonstrate that a quantitative RT-PCR assay designed to recognize an engineered intron splice site in the 5'UTR of expression constructs effectively measures the expression level of in vivo-delivered gene therapeutics.

Characterization and in vivo testing of a heterogeneous cationic lipid-DNA formulation.

PURPOSE: To identify characteristics of lipid-DNA complexes that correlate with in vivo expression data. METHODS: DOTIM:cholesterol liposomes (1:1 mole ratio) and DNA expressing chloramphenicol acetyl transferase (CAT) were complexed at a 4.2:1 mass ratio (cationic lipid:DNA). Complexes were fractionated by density gradient centrifugation. analyzed for particle size and zeta potential and quantitated using HPLC methods. The unfractionated complexes, "purified" fractions of the complexes, and purified complexes supplemented with liposomes were administered to mice by intravenous injection (i.v.) and intratracheal instillation (i.t.) and their ability to express gene product was assessed. RESULTS: Centrifugation separated two distinct populations within complexes one consisting of free liposomes and the other of lipid complexed with DNA. The vesicle diameter and zeta potential among separated fractions varied from 113 to 354 nm. and + 24 to + 34 mV respectively. Re-centrifugation of the 'purified' fractions containing the lipid-DNA population produced a single band. CAT expression in lung tissue 24 hr post-i.v. was observed with the unfractionated complex, but not the purified form. Some activity was 'restored' with the liposome-supplemented complexes. In contrast, the same series of complexes administered by i.t. resulted in no significant difference in lung expression (p=0.16 ANOVA). CONCLUSIONS: An uncomplexed liposome population exists within DOTIM:cholesterol-DNA complexes that influences the expression of complexes administered i.v. but not i.t..

Metastasizing neuroblastomas in mice transgenic for simian virus 40 large T (SV40T) under the olfactory marker protein gene promoter.

The olfactory marker protein (OMP) is a M(r) 19,000 polypeptide originally considered a selective marker for differentiated olfactory receptor neurons. In an attempt to induce neoplastic proliferation in the olfactory cells, we made mice transgenic for the simian virus 40 large T-antigen gene linked to the OMP gene promoter. Four independent lines of transgenic mice were established. Despite a high expression of the transgene in the olfactory receptor neurons, no evidence of tumor growth was observed. Instead, starting from an age of 4 months, animals of all four lines presented with highly metastatic tumors originating in the adrenal medullas or sympathetic ganglia. The histological, ultrastructural, and immunohistochemical features of the tumors were identical to those of human infant neuroblastoma. Five independent neuroblastoma cell lines were established from tumors of different transgenic animals. All cell lines constitutively express the endogenous OMP gene. The transgene product, simian virus 40 large T-antigen, associates with the product of the anti-oncogene p53 in these cell lines. This transgene system not only offers a biologically faithful model for investigations on the pathogenesis of neuroblastoma, the most common solid neoplasia of infancy, it also raises intriguing questions about the role of the OMP gene for the differentiation of the sympathetic neurons.

Chromosomal location and isoform analysis of mouse Fc epsilon RII/CD23.

The gene for the mouse low affinity receptor for IgE (Fc epsilon RII, also known as CD23) was mapped on Chromosome (Chr) 8 proximal to Plat. This gene, symbolized Fcer2 (formerly Fce2) resides in a region of Chr 8 with linkage homology with human chromosomes 8 and 19. The mouse Fc epsilon RII was examined for the presence of alternate N-terminal forms such as seen in humans. An antisense RNA probe was prepared from the 5' end of the cDNA through the first 660 bp of the cDNA and was used to analyze message from Fc epsilon RII+ B cells and B cell hybridomas both before and after treatment with interleukin 4 (IL-4). Using RNase protection analysis, a major 640 bp band corresponding to the full length probe was seen, even after activation of the cells with LPS in the presence of IL-4, which is known to give high expression levels of the Fc epsilon RII. This result suggests that the mouse does not produce significant levels of an alternate IL-4 inducible Fc epsilon RII, as seen in man, and this may explain the more restricted cell lineage expression of the Fc epsilon RII in the mouse.

Qa-2 expression in the adult murine thymus. A unique marker for a mature thymic subset.

The MHC Ag, Qa-2, is expressed on all peripheral T cells, a subset of bone marrow cells, and to a lesser extent on B cells. The Qa-2 Ag is also expressed on 5 to 6% of normal adult murine thymocytes. Through the use of flow cytometry, counterflow centrifugal elutriation and acridine orange staining, we have analyzed the cell surface phenotype, cell size, and cell cycle status of this thymic population. Our studies indicate that Qa-2+ thymocytes are large, non-mitotic, G1 cells which have the cell surface phenotype of CD5+, CD3+, J11dLO and lack receptors for peanut agglutinin. This population can be further subdivided into three categories; CD4+/CD8-, CD4-/CD8+, and CD4-/CD8-. These data indicate that Qa-2 surface expression can only be detected on thymocytes in the final stages of differentiation. The Qa-2 Ag can be used as a cell surface marker to identify a unique subset of mature thymocytes.

Qa gene expression: biosynthesis and secretion of Qa-2 molecules in activated T cells.

The biosynthesis and expression of the tissue-specific class I molecule Qa-2 have been studied in resting and activated T-cell populations. Polyclonal activation of T lymphocytes induces a 3- to 4-fold increase in the biosynthesis of Qa-2 molecules but no increase in cell-surface levels. Analysis of the biosynthetic pathway of the Qa-2 molecule in activated lymphocytes reveals that approximately equal to 70% of the newly synthesized Qa-2 molecules are secreted as soluble molecules. In resting-cell populations, Qa-2 remains entirely cell-associated. This process is unique to the Qa-2 molecule, since other class I molecules (e.g., H-2Kb and H-2Db) synthesized by activated cells remain cell-associated. The possibility that the secreted Qa-2 molecule is the product of a new Qa gene or an alternatively spliced mRNA is considered. These results indicate that the Qa-2 molecules may not just function as a cell-surface recognition structure but also may serve a role as a soluble factor synthesized by activated lymphoid cell populations.