Abstract
Background
Human immunodeficiency virus type 1 (HIV‐1)‐based lentiviral vectors can infect dividing and non‐dividing cells. The purpose of this study was to investigate the efficacy of lentiviral transfer of the green fluorescent protein (eGFP) gene into post‐mitotic quail skeletal muscle and into fibers undergoing hypertrophy and hyperplasia via addition of satellite cell nuclei.
Methods
A vesicular stomatitis virus G‐protein (pMFG)‐pseudotyped lentiviral vector encoding the eGFP gene coding sequence, flanked by two long terminal repeats (LTRs) (pHR‐CMV‐eGFP‐Wsin‐18) and an optional plasmid (pRSVrev), was generated in 293T cells by means of the three‐plasmid system. The resulting lentiviral vector, LtV/eGFP, was injected in vivo into the patagialis muscles of young (6 weeks of age) and senescent (100 weeks of age) Japanese quail. Polybrene (10 µg/muscle) was injected in the right patagialis muscles of some birds. A weight was attached to the left wing of birds immediately following LtV/eGFP injection for 7, 14 or 21 days to induce hypertrophy in the patagialis muscle via satellite cell activation. A time‐released bromodeoxyuridine (BrdU) pellet was implanted in some birds to identify satellite cells that had proliferated during stretch‐overload. Control and experimental muscles were examined 7, 14 or 21 days following injection and wing loading.
Results
Seven days post‐injection proved to be insufficient to visualize and quantify the eGFP signal. EGFP‐positive fibers were evident 14 and 21 days after injection of LtV/eGFP. As expected, wing weighting resulted in hypertrophy of the patagialis of senescent and young birds and an increase in collagen and other connective tissue in muscles of old birds. Nevertheless, this did not interfere with expression levels or intensity of the eGFP vector because the area of eGFP‐positive fibers normalized to total muscle area was similar in control and overloaded muscles, and in muscles that were injected with the lentivirus vector but not loaded. Polybrene improved eGFP expression by 41% as compared with muscles that received only the lentivirus.
Conclusions
These findings provide the first evidence that this lentivirus vector is an efficient tool for gene transfer and expression in mature (non‐growing) fibers of adult birds, sarcopenic fibers of senescent birds and hypertrophying skeletal muscle fibers of both young adult and old birds. This system could be useful for tissue engineering, or treating aging‐associated muscle‐wasting diseases such as sarcopenia, especially when exercise/overload are added to gene therapy approaches to combat muscle loss with aging. Copyright © 2004 John Wiley & Sons, Ltd.