Generation of SV40-transformed rabbit tracheal-epithelial-cell-derived blastocyst by somatic cell nuclear transfer.

The prospect of developing large animal models for the study of inherited diseases, such as cystic fibrosis (CF), through somatic cell nuclear transfer (SCNT) has opened up new opportunities for enhancing our understanding of disease pathology and for identifying new therapies. Thus, the development of species-specific in vitro cell systems that will provide broader insight into organ- and cell-type-specific functions relevant to the pathology of the disease is crucial. Studies have been undertaken to establish transformed rabbit airway epithelial cell lines that display differentiated features characteristic of the primary airway epithelium. This study describes the successful establishment and characterization of two SV40-transformed rabbit tracheal epithelial cell lines. These cell lines, 5RTEo- and 9RTEo-, express the CF transmembrane conductance regulator (CFTR) gene, retain epithelial-specific differentiated morphology and show CFTR-based cAMP-dependent Cl(-) ion transport across the apical membrane of a confluent monolayer. Immunocytochemical analysis indicates the presence of airway cytokeratins and tight-junction proteins in the 9RTEo- cell line after multiple generations. However, the tight junctions appear to diminish in their efficacy in both cell lines after at least 100 generations. Initial SCNT studies with the 9RTEo- cells have revealed that SV40-transformed rabbit airway epithelial donor cells can be used to generate blastocysts. These cell systems provide valuable models for studying the developmental and metabolic modulation of CFTR gene expression and rabbit airway epithelial cell biology.

Targeted gene correction with 5' acridine-oligonucleotide conjugates.

Single-stranded oligonucleotides (SSOs) mediate gene repair of punctual chromosomal mutations at a low frequency. We hypothesized that enhancement of DNA binding affinity of SSOs by intercalating agents may increase the number of corrected cells. Several biochemical modifications of SSOs were tested for their capability to correct a chromosomally integrated and mutated GFP reporter gene in human 293 cells. SSOs of 25 nucleotide length conjugated with acridine at their 5' end increased the efficiency of gene correction up to 10-fold compared to nonmodified SSOs. Acridine and psoralen conjugates were both evaluated, and acridine-modified SSOs were the most effective. Conjugation with acridine at the 3' end of the SSO inhibited gene correction, whereas flanking the SSO by acridine on both sides provided an intermediate level of correction. These results suggest that increasing the stability of hybridization between SSO and its target without hampering a 3' extension improves gene targeting, in agreement with the "annealing-integration" model of DNA repair.

Improving gene replacement by intracellular formation of linear homologous DNA.

BACKGROUND: Gene targeting is a potential tool for gene therapy but is limited by the low rate of homologous recombination. Using highly homologous linear DNA improves gene targeting frequency but requires microinjection into nuclear cells to be effective. Because transfection of circular DNA is more efficient than transfection of linear DNA and adaptable to viral vectors, we developed a system for the intracellular release of linear fragments from circular plasmids. METHODS: Only one cutting site inside the "donor" DNA was not convenient because it led to integration of exogenous sequences into the target. So we constructed several "donor" plasmids containing the homologous sequences flanked by two I-Sce I recognition sites. Expression of I-Sce I allowed intracellular delivery of "ends-out" (replacement) vectors. We compared the efficiency of different constructions to correct a mutated gfp target. RESULTS: Co-transfection of "donor" plasmids and an I-Sce I expression vector into CHO cells enhanced the correction of an extrachromosomal mutated gfp target by at least 10 times. Maximum correction was observed with the greatest homology size and maximum effect of I-Sce I was obtained when the long hemi-sites of the duplicated I-Sce I sites were contiguous to the homologous sequence. Unexpectedly, the reverse orientation of I-Sce I sites provided little or no effect, probably due to the asymmetrical activity of the I-Sce I meganuclease. CONCLUSIONS: Releasing homologous DNA fragments with I-Sce I enhances gene replacement. This work provides the basis for the future design of viral vectors for gene replacement.

A new method (GOREC) for directed mutagenesis and gene repair by homologous recombination.

Directed mutagenesis in mammalian cells has been the focus of intense research because of its promising application for gene correction and engineering. Both natural and modified oligonucleotides (ODN), RNA-DNA chimeric oligonucleotide (RDO) and small fragment DNA (SFHR), as well as vector DNA were used for promoting homologous replacement with varying success. It was recently shown that a triple helix-forming oligonucleotide (TFO) tethered to an oligonucleotide (donor DNA) can enhance mutagenesis by homologous recombination in cells. The basic idea is to accelerate homology search by oligonucleotide-directed triple helix formation in the vicinity of the target site for donor DNA. Here we describe a new method named GOREC (guided homologous recombination) which shares similar gene targeting, but has notable difference in the concept with the previous method. It is made of a homing device (TFO) and a donor DNA for effecting distinct functions. They are linked together by non-covalent or covalent interaction. This modular concept allows guidance of either an oligonucleotide (ODN, RDO) or a small DNA fragment to the target site for homologous replacement. Therefore, the triple helix site can be hundreds of base pairs away from the target site. An episomal assay for proof-of-principle study will be presented and discussed.

Stimulation of RecA-mediated D-loop formation by oligonucleotide-directed triple-helix formation: guided homologous recombination (GOREC).

Oligonucleotide-directed triple helix formation provides an elegant rational basis for gene-specific DNA targeting and has been widely used to interfere with gene expression ("antigene" strategies) and as a molecular tool for biological studies. Various strategies have been developed to introduce sequence modifications in genomes. However, the low efficiency of the overall process in eucaryotic cells impairs efficient recovery of recombinant genomes. Since one limiting step in homologous recombination is the targeting to the homologous sequence, we have tested the contribution of an oligonucleotide-directed triple helix formation on the RecA-dependent association of an oligonucleotide and its homologous target on duplex DNA (D-loop formation). For this study, the recombinant ssDNA fragment was noncovalently associated to a triple helix-forming oligonucleotide. The physicochemical and biochemical characteristics of the triple helix and D-loop structures formed by the complex molecules in the presence or in the absence of RecA protein were determined. We have demonstrated that the triple helix-forming oligonucleotide increases the efficiency of D-loop formation and the RecA protein speeds up also the triple helix formation. The so-called "GOREC" (for guided homologous recombination) approach can be developed as a novel tool to improve the efficiency of directed mutagenesis and gene alteration in living organisms.

Incorporation of a novel nucleobase allows stable oligonucleotide-directed triple helix formation at the target sequence containing a purine.pyrimidine interruption.

Thermal denaturation experiments have established that an oligonucleotide incorporating the artificial nucleobase S, does form a stable triplex with a double stranded DNA which exhibits a pyrimidine interruption within the oligopurine sequence.

DNA triplex structures are stabilized by the incorporation of 3'-endo blocked pyrimidine nucleosides in the Hoogsteen strand.

A short route to pyrimidine locked nucleosides has been developed for their incorporation in triplex forming oligonucleotides (TFO). Compared to oligonucleotides built with standard nucleosides, the modified TFOs containing 3'-endo blocked residues formed, with their corresponding DNA duplexes, more stable triple helix systems, an effect which might be ascribed to the 3'-endo pucker of the modified nucleoside residues.