title: Expression levels of the PiT-2 receptor explain, in part, the gestational age-dependent alterations in transduction efficiency after in utero retroviral-mediated gene transfer.
abstract: BACKGROUND
  A fundamental obstacle to using retroviral-mediated gene transfer (GT) to treat human diseases is the relatively low transduction levels that have been achieved in clinically relevant human cells. We previously showed that performing GT in utero overcomes this obstacle and results in significant levels of transduction within multiple fetal organs, with different tissues exhibiting optimal transduction at different developmental stages. We undertook the present study aiming to elucidate the mechanism for this age-dependent transduction, testing the two factors that we hypothesized could be responsible: (i) the proliferative status of the tissue at the time of GT and (ii) the expression level of the amphotropic PiT-2 receptor.
  
  
  METHODS
  Immunofluorescence was performed on tissues from sheep of varying developmental stages to assess the proliferative status of the predominant cells within each organ as a function of age. After developing an enzyme-linked immunosorbent assay (ELISA) and a quantitative reverse transcription chain reaction (qRT-PCR) assay, we then quantified PiT-2 expression at the protein and mRNA levels, respectively.
  
  
  RESULTS
  The results obtained indicate that the proliferative status of organs at the time of fetal GT is not the major determinant governing transduction efficiency. By contrast, our ELISA and qRT-PCR analyses demonstrated that PiT-2 mRNA and protein levels vary with gestational age, correlating with the observed differences in transduction efficiency.
  
  
  CONCLUSIONS
  The findings of the present study explain the age-related differences that we previously observed in transduction efficiency after in utero GT. They also suggest it may be possible to achieve relatively selective GT to specific tissues by performing in utero GT when levels of PiT-2 are maximal in the desired target organ.
authors.affiliation: Department of Animal Biotechnology, University of Nevada, Reno, NV, USA.
authors.forename: Ferhat
authors.initials: F
authors.lastname: Ozturk
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Park
authors.forename: Joseph
authors.initials: J
authors.lastname: Tellez
authors.forename: Evan
authors.initials: E
authors.lastname: Colletti
authors.forename: Maribeth V
authors.initials: MV
authors.lastname: Eiden
authors.forename: Graça
authors.initials: G
authors.lastname: Almeida-Porada
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Porada
mesh_terms: D000818:Animals; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005333:Fetus / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D005865:Gestational Age; D011991:Receptors, Virus / Q000378:metabolism*; D012190:Retroviridae; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012756:Sheep; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: D011991:Receptors, Virus; C067981:leukemia virus receptor, gibbon ape