Although the primary function of U1 snRNA is to define the 5 donor site of an intron, it can also block the accumulation of a specific RNA transcript when it binds to a donor sequence within its terminal exon. This work was initiated to investigate if this property of U1 snRNA could be exploited as an effective method for inactivating any target gene. The initial 10-bp segment of U1 snRNA, which is complementary to the 5 donor sequence, was modified to recognize various target mRNAs (

Marrow stromal cells (MSC) and neonatal calvarial cells have the potential to differentiate and express markers of mature osteoblasts. Furthermore, MSCs can generate multiple differentiated connective tissue phenotypes. These properties and their ability to be expanded ex vivo make them good models for ex vivo gene therapy. In this study we examined the ability of vesicular stomatitis virus (VSV-G) pseudotyped retroviral vectors to transduce osteoprogenitor cells derived from bone marrow and from neonatal calvaria. Retrovectors encoding either beta-galactosidase or green fluorescent protein (eGFP) were used for transduction of primary murine marrow stromal and primary neonatal calvarial cell cultures. High infection efficiency was demonstrated by fluorescence-activated cell analysis when GFP was used as a marker or by estimating the number of beta-galactosidase-positive cells. Expression of markers of differentiated bone cells, including Col1a1, bone sialoprotein, and osteocalcin mRNA and alkaline phosphatase activity was not impaired by retroviral transduction. Our data suggest that VSV-G pseudotypes retroviral vectors are suitable for introducing genes into osteoprogenitor cells without affecting osteoprogenitor lineage progression.

The modified U1 snRNA gene can suppress expression of a target transgene. In the present study, its potential utility to inhibit a dominant negative/gain of function mutation is explored. Using a green fluorescent protein (GFP) target gene, inhibition was achieved in all cells transduced with U1antiGFP directed at multiple sites within GFP. Using a chloramphenicol acetyltransferase (CAT) target gene, inhibition was not increased by increasing the hybridization domain from 10 to 16 bp or when a site in an upstream exon or intron was targeted. To determine if a U1 anti-target design could discriminate between two transcripts that differ by a 1-2 bp mismatch, GFPtpz and GFPsaph were chosen as targets because they share sequence homology except for three regions where a 1, 2 or 3 bp mismatch exists. The results demonstrated that U1antiGFP correctly reduced its cognate GFP expression by >90% and therefore U1 anti-target constructs are able to discriminate a 1 or 2 bp mismatch in their target mRNA. Thus, these U1 anti-target constructs may be effective in a strategy of somatic gene therapy for a dominant negative/gain of function mutation due to the discreteness of its discrimination. It may complement other anti-target strategies to reduce the cellular load of a mutant transcript.

Fibroblast growth factor-2 (FGF-2) is made by osteoblasts and modulates their function. There are high molecular weight (HMW) protein isoforms of FGF-2 that have nuclear localization sequences and a low molecular weight (LMW) 18 kDa FGF-2 protein that is exported from cells. Since FGF-2 is a trophic factor and potent mitogen for osteoblasts, the goal of this study was to utilize targeted overexpression of FGF-2 as a novel means of assessing different FGF-2 isoforms on osteoblastic cell viability and proliferation. Either LMW or HMW human Fgf2 cDNAs were cloned downstream of 3.6 kb alpha1(I)-collagen 5' regulatory elements (Col 3.6). A set of expression vectors, called Col3.6-Fgf2 isoforms-IRES-GFPsaph, capable of concurrently overexpressing either LMW or HMW FGF-2 isoforms concomitant with GFPsaph from a single bicistronic mRNA were built. Viable cell number in ROS 17/2.8 cells stably transfected with Vector (Col3.6-IRES-GFPsaph) versus each of the Col3.6-Fgf2-IRES-GFPsaph constructs were compared. In the presence of 1 or 10% serum, DNA synthesis was increased in cells expressing any isoform of FGF-2 compared with vector. However, cells transfected with HMW isoform had augmented DNA synthesis in 1 or 10% serum compared with cells expressing either ALL or LMW FGF-2 isoforms. A neutralizing FGF-2 antibody significantly reduced the mitogenic response in cells harboring ALL or the LMW FGF-2 isoforms but did not block the mitogenic effect of cells harboring the HMW isoforms. In summary, overexpression of any isoform of FGF-2 protein increased viable cell number and OB proliferation in the presence of low or high concentrations of serum. However, the HMW/nuclear isoforms preferentially mediate augmented OB proliferation. We conclude that differential expression of FGF-2 proteins isoforms is important in modulating OB function.