Biomedical subjects

J S Latendresse

Publications and source records attributed to J S Latendresse.

5 recordsLinked to original sources

PubMed1997

High expression of naked plasmid DNA in muscles of young rodents.

There is a time window at 2 weeks of age for achieving very high levels of foreign gene expression from the intramuscular injection of naked plasmid DNA in mice and rats. The highest expression, over 1 microg of luciferase protein/muscle, was obtained in Balb/C mice using constructs containing the CMV promoter, a chimeric intron and the luc+ luciferase gene. Approximately 50% of the myofibers were intensely blue following the intramuscular injection of a beta-galactosidase expression vector in 2 week old Balb/C mice. The effects of age, mouse strain and construct were multiplicative, resulting in >1000-fold greater luciferase and approximately 20-fold more beta-galactosidase-positive cells. These high levels of expression were unstable and were not observed in larger animals (dog, rhesus monkey). These results indicate that enormous levels of foreign gene expression can be obtained in muscle with naked DNA in vivo and will enable the temporary effects of gene function and expression in rodent muscle to be expeditiously studied.

Age Factors↗

PubMed1995

A plasmid-based self-amplifying Sindbis virus vector.

Sindbis virus was used as a self-amplifying eukaryotic expression vector. A recombinant cDNA genome of this (+)-strand RNA virus was placed under the transcriptional control of a Rous sarcoma virus LTR (RSV) promoter. Transfection of this plasmid construct into mammalian cell lines (3T3, HepG2, and 293 cells) resulted in expression of the luciferase reporter gene. High-expression levels were also measured after transfection into primary rat myoblasts. In differentiated myotubes, expression levels generated by the Sindbis virus vector were up to 200 times higher than those obtained with a conventional RSV expression vector. In vivo expression was detected after injection of plasmid DNA into mouse quadriceps. In vivo expression was transient and undetectable by day 16. This self-amplifying expression vector can be used for generating high-level expression of transgenes in vitro and in vivo. Its transient nature in vivo could allow for safe, short-term delivery of gene products in gene therapy protocols. It should facilitate the study of Sindbis and other RNA viruses.

3T3 Cells↗

PubMed1995

Expression of deletion-containing dystrophins in mdx muscle: implications for gene therapy and dystrophin function.

The expression of full-length dystrophin and various dystrophin deletion mutants was monitored in mdx mouse muscle after intramuscular injection of dystrophin-encoding plasmid DNAs. Recombinant dystrophin proteins, including those lacking either the amino terminus, carboxyl terminus, or most of the central rod domain, showed localization to the plasma membrane. This suggests that there are multiple attachment sites for dystrophin to the plasma membrane. Only those constructs containing the carboxyl terminus were able to stabilize dystrophin-associated proteins (DAP) at the membrane, consistent with other studies that suggest that this domain is critical to DAP binding. Colocalization with DAP was not necessary for membrane localization of the various dystrophin molecules. However, stabilization and co-localization of the DAP did seem to be a prerequisite for expression and/or stabilization of mutant dystrophins beyond 1 wk and these same criteria seemed important for mitigating the histopathological consequences of dystrophin deficiency.

Animals↗

PubMed1994

Pharmacological enhancement of in vivo foreign gene expression in muscle.

Intramuscular injection of naked plasmid DNA provides a means for gene transfer and expression in striated muscle. In this study, the effects of treating muscle with normal saline, etidocaine, mepivacaine, acetic anhydride, sodium bicarbonate, Notechis scutatus venom, cardiotoxin and bupivacaine before plasmid DNA injection on foreign gene expression were evaluated. Dose dependence, strain and species specificity, the time interval between pharmacological agent and plasmid DNA injection, the stability of gene expression and the fate of the injected plasmid DNA were studied using reporter gene expression, by histological examination and semi-quantitative polymerase chain reaction. Of the various agents tested, the best enhancement of foreign gene expression occurred in muscle treated with 0.75% bupivacaine five to seven days before plasmid DNA injection. Rat and mouse quadriceps muscle treated with 0.75% bupivacaine had levels of luciferase activity four- to 40-times greater than non-bupivacaine-treated muscle. Also, beta-galactosidase expressing myofibers were observed throughout the length of the muscle in samples treated with 0.75% bupivacaine before reporter gene injection. Muscle treated with 0.75% bupivacaine fully recovered from the degeneration caused by its injection with no long-term effects histologically. The heightened level of reporter gene expression persisted in 0.75% bupivacaine-treated muscle for one month, but decreased to that of non-bupivacaine-treated muscle by two months after plasmid DNA injection. Enhancement of foreign gene expression may be particularly advantageous in vaccination protocols employing intramuscular plasmid injection.

Acetic Anhydrides↗

PubMed1993

Dystrophin expression improves myofiber survival in mdx muscle following intramuscular plasmid DNA injection.

Expression of Becker-like and full-length human dystrophins was stable for at least 6 months in mdx mouse muscle following intramuscular plasmid DNA injection. Intramuscular injection of a single plasmid DNA encoding both luciferase and dystrophin resulted in stable luciferase expression for at least 2 months in mdx muscle, whereas injection of plasmid DNA encoding only luciferase did not result in stable luciferase expression. These results suggest that expression of either full-length or Becker-like dystrophins protects mdx mouse myofibers from degeneration.

Animals↗