Showing papers by "Transgene SA published in 1992"

Expression of the human cystic fibrosis transmembrane conductance regulator gene in the mouse lung after in vivo intratracheal plasmid-mediated gene transfer

Abstract: As an approach to gene therapy for the respiratory manifestations of cystic fibrosis (CF), in vivo plasmid-mediated direct transfer of the normal CF transmembrane conductance regulator (CFTR) gene to the airway epithelium was investigated in mice. To evaluate the feasibility of this strategy, pRSVL, a plasmid composed of a firefly luciferase gene driven by the Rous sarcoma virus long terminal repeat (RSV-LTR), along with cationic liposomes was instilled into the trachea of C57BI/6NCR mice. With administration of 200-400 micrograms plasmid DNA, luciferase expression could be detected in the mouse lung homogenates for at least 4 wk. With this background, a CFTR expression plasmid vector (pRSVCFTR) constructed by replacing the luciferase cDNA from pRSVL with the normal human CFTR cDNA was evaluated in vivo in mice. Intratracheal instillation of pRSVCFTR with cationic liposomes followed by analysis of mouse lung RNA by polymerase chain reaction amplification (after conversion of mRNA to cDNA) using a RSV-LTR specific sense primer and a human CFTR-specific antisense primer demonstrated human CFTR mRNA transcripts from one day to 4 wk after instillation. Further, in vivo evaluation of beta-galactosidase activity after intratracheal administration of an E. coli lacZ gene expression plasmid vector directed by the cytomegalovirus promoter (pCMV beta) demonstrated that the airway epithelium was the major target of transfer and expression of the exogenous gene. These observations demonstrate successful plasmid-mediated gene transfer to the airway epithelium in vivo. This strategy may be feasible as a form of gene therapy to prevent the pulmonary manifestations of CF.

Rapid mycobacterial plasmid analysis by electroduction between Mycobacterium spp. and Escherichia coli.

Abstract: In recent years, the prospect of using Mycobacterium bovis BCG as a live vaccine carrier for foreign antigens has prompted a rapid evolution of the molecular biology of Mycobacteria. This progress was considerably facilitated by the development of the electroporation technique allowing high levels of transformation rates of this organism (1). Nowadays, Escherichia coliMycobacteria shuttle plasmids are commonly used to take full advantage of the molecular biological tools developed in E.coli, before transferring the final constructions into the mycobacterial species. However, plasmid analysis of mycobacterial transformants still presents major difficulties. The slow growth rate of cell cultures impedes rapid analysis of recombinant bacteria (see Table 1). Moreover, yields of extracted plasmid DNA are usually poor, essentially due to the high resistance of Mycobacteria to standard cell lysis in addition to generally low plasmid copy numbers. We report here a simple technique allowing a significantly faster analysis of plasmid DNA from mycobacterial transformants. The method is based on two basic features, i.e. the inherent ability of shuttle plasmids to replicate in at least two distant bacterial species, and the fact that DNA molecules may be introduced or released from a bacterial cell submitted to electroporation. Indeed, both inter(2, 3) and intraspecies (4) DNA transfers have already been demonstrated during electroporation. Interspecies transfer usually requires two pulses. We demonstrate direct plasmid transfer between Mycobacterium smegmatis and M.bovis BCG, and E.coli, two very distant bacterial genera, following a single electrical pulse. M. smegmatis m c 2 ^ and M.bovis BCG 1173P2 were transformed (1) with the shuttle plasmid pRR3 (5) and plated on Middlebrook 7H10 agar supplemented with Middlebrook ADC Enrichment (Difco) + 15 /tg/ml kanamycin. Visible colonies were obtained after incubation for 4 days for M. smegmatis or 14 days for M.bovis BCG at 37 °C. Twelve growing colonies were picked and mixed with 20 fi\\ of ice-cold 10% glycerol. The tubes were briefly vortexed before placing on ice for 10 min. These mixtures were added to E.coli electro-competent cells (at a concentration of 10'° cells per ml) prepared as described previously (6). After a single electric pulse using parameters optimal for E.coli electrotransformation (135 W, 2500 V, 45 /iF on the Cellject apparatus, Eurogentech), cells were incubated for 1 hour at 37°C before plating on LB medium supplemented with 25 /ig/ml of kanamycin. Resistant E.coli cells appeared after 12 — 16 hours at 37 °C and plasmid DNA was extracted and analysed the next day using standard procedures. Direct DNA transfer between BCG and E.coli resulted in 10-100 transformants per M.bovis colony. This technique was even more efficient between M.smegmatis and E.coli; a mean of 10 transformants obtained per M.smegmatis colony. Moreover, contamination risks inherent to long term mycobacterial cultures are avoided, as are the laborious and low yield DNA extractions from this organism. This protocol should be applicable to general use, especially for slow-growing or fastidious bacteria or for those that are resistant to standard cell lysis procedures.

Rna delivery vector

Abstract: An RNA delivery vector including a liposome and at least one RNA fragment coding for an antigenic determinant which characterizes a tumor or a pathogenic agent, said at least one RNA fragment being encapsulated in said liposome.

Process for the purification of a highly glycosylated protein

Abstract: L'invention propose un procede pour purifier une proteine fortement glycosylee a partir d'une preparation brute qui comprend l'acte (i) d'ajouter a ladite preparation un ion metallique bivalent en quantite suffisante de maniere a former un melange qui precipite et (ii), apres precipitation, de recolter ladite proteine dans le surnageant du melange. The invention provides a method for purifying a highly glycosylated protein from a crude preparation which comprises the action (i) adding to said preparation a divalent metal ion in an amount sufficient to form a mixture which precipitates and ( ii) after precipitation, of harvesting the said protein from the mixture supernatant.

Novel cell lines derived from transgenic mice expressing recombinant human proteins. Transgenic hepatoma-derived cell lines.

Abstract: We have used transgenic mouse technology to establish immortalized hepatoma cell lines stably secreting heterologous proteins, such as human α1-antitrypsin and human factor IX. Hepatocyte-specific regulatory DNA sequences were used to target both the expression of anonc gene and the gene coding for the human protein to the liver of transgenic mice which eventually developed hepatocellular carcinomas. Tumour cells were subsequently established as permanent cell lines, which maintained a differentiated phenotype under specific culture conditions, being capable of producing biologically active and correctly processed human α1-antitrypsin and factor IX. Moreover, a preliminary analysis has shown that certain cell lines express elevated total cytochrome P450 activity. These cells could therefore represent a useful alternative to the use of animals or primary cultures in drug safety testing.