title: Evaluation of biolistic gene transfer methods in vivo using non-invasive bioluminescent imaging techniques.
abstract: BACKGROUND
  Gene therapy continues to hold great potential for treating many different types of disease and dysfunction. Safe and efficient techniques for gene transfer and expression in vivo are needed to enable gene therapeutic strategies to be effective in patients. Currently, the most commonly used methods employ replication-defective viral vectors for gene transfer, while physical gene transfer methods such as biolistic-mediated ("gene-gun") delivery to target tissues have not been as extensively explored. In the present study, we evaluated the efficacy of biolistic gene transfer techniques in vivo using non-invasive bioluminescent imaging (BLI) methods.
  
  
  RESULTS
  Plasmid DNA carrying the firefly luciferase (LUC) reporter gene under the control of the human Cytomegalovirus (CMV) promoter/enhancer was transfected into mouse skin and liver using biolistic methods. The plasmids were coupled to gold microspheres (1 μm diameter) using different DNA Loading Ratios (DLRs), and "shot" into target tissues using a helium-driven gene gun. The optimal DLR was found to be in the range of 4-10. Bioluminescence was measured using an In Vivo Imaging System (IVIS-50) at various time-points following transfer. Biolistic gene transfer to mouse skin produced peak reporter gene expression one day after transfer. Expression remained detectable through four days, but declined to undetectable levels by six days following gene transfer. Maximum depth of tissue penetration following biolistic transfer to abdominal skin was 200-300 μm. Similarly, biolistic gene transfer to mouse liver in vivo also produced peak early expression followed by a decline over time. In contrast to skin, however, liver expression of the reporter gene was relatively stable 4-8 days post-biolistic gene transfer, and remained detectable for nearly two weeks.
  
  
  CONCLUSIONS
  The use of bioluminescence imaging techniques enabled efficient evaluation of reporter gene expression in vivo. Our results demonstrate that different tissues show different expression kinetics following gene transfer of the same reporter plasmid to different mouse tissues in vivo. We evaluated superficial (skin) and abdominal organ (liver) targets, and found that reporter gene expression peaked within the first two days post-transfer in each case, but declined most rapidly in the skin (3-4 days) compared to liver (10-14 days). This information is essential for designing effective gene therapy strategies in different target tissues.
authors.affiliation: Burnett School of Biomedical Sciences, University of Central Florida College of Medicine, Orlando, FL 32827, USA.
authors.forename: Jixiang
authors.initials: J
authors.lastname: Xia
authors.forename: Angela
authors.initials: A
authors.lastname: Martinez
authors.forename: Henry
authors.initials: H
authors.lastname: Daniell
authors.forename: Steven N
authors.initials: SN
authors.lastname: Ebert
mesh_terms: D000818:Animals; D019470:Biolistics / Q000379:methods*; D003587:Cytomegalovirus / Q000235:genetics; D017930:Genes, Reporter; D006046:Gold / Q000737:chemistry; D006801:Humans; D008099:Liver / Q000378:metabolism; D049409:Luciferases, Firefly / Q000235:genetics; D008163:Luminescent Measurements / Q000379:methods*; D051379:Mice; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012867:Skin / Q000378:metabolism
chemical_list: D006046:Gold; D049409:Luciferases, Firefly