title: Successful nucleofection of rat adipose-derived stroma cells with Ambystoma mexicanum epidermal lipoxygenase (AmbLOXe).
abstract: BACKGROUND
  Adipose-derived stroma cells (ASCs) are attractive cells for cell-based gene therapy but are generally difficult to transfect. Nucleofection has proven to be an efficient method for transfection of primary cells. Therefore, we used this technique to transfect ASCs with a vector encoding for Ambystoma mexicanum epidermal lipoxygenase (AmbLOXe) which is a promising bioactive enzyme in regenerative processes. Thereby, we thought to even further increase the large regenerative potential of the ASCs.
  
  
  METHODS
  ASCs were isolated from the inguinal fat pad of Lewis rats and were subsequently transfected in passage 1 using Nucleofector® 2b and the hMSC Nucleofector kit. Transfection efficiency was determined measuring co-transfected green fluorescent protein (GFP) in a flow cytometer and gene expression in transfected cells was detected by reverse transcription polymerase chain reaction (RT-PCR). Moreover, cell migration was assessed using a scratch assay and results were tested for statistical significance with ANOVA followed by Bonferroni's post hoc test.
  
  
  RESULTS
  High initial transfection rates were achieved with an average of 79.8 ± 2.82% of GFP positive cells although longer cultivation periods reduced the number of positive cells to below 5% after four passages. Although successful production of AmbLOXe transcript could be proven the gene product had no measureable effect on cell migration.
  
  
  CONCLUSIONS
  Our study demonstrates the feasibility of ASCs to serve as a vehicle of AmbLOXe transport for gene therapeutic purposes in regenerative medicine. One potential field of applications could be peripheral nerve injuries.
authors.forename: Angela
authors.initials: A
authors.lastname: Fülbier
authors.forename: Reinhild
authors.initials: R
authors.lastname: Schnabel
authors.forename: Stefanie
authors.initials: S
authors.lastname: Michael
authors.forename: Peter M
authors.initials: PM
authors.lastname: Vogt
authors.forename: Sarah
authors.initials: S
authors.lastname: Strauß
authors.forename: Kerstin
authors.initials: K
authors.lastname: Reimers
authors.forename: Christine
authors.initials: C
authors.lastname: Radtke
mesh_terms: D000273:Adipose Tissue / Q000166:cytology / Q000201:enzymology / Q000502:physiology*; D000558:Ambystoma mexicanum / Q000235:genetics / Q000378:metabolism; D000818:Animals; D015870:Gene Expression; D008084:Lipoxygenase / Q000096:biosynthesis / Q000235:genetics*; D008297:Male; D051381:Rats; D011917:Rats, Inbred Lew; D014162:Transfection / Q000379:methods*
chemical_list: D008084:Lipoxygenase