title: Functional promoter testing using a modified lentiviral transfer vector.
abstract: OBJECTIVE
  The importance of retinal glial cells in the maintenance of retinal health and in retinal degenerations has not been fully explored. Several groups have suggested that secretion of neurotrophic proteins from the retina's primary glial cell type, the Müller cell, holds promise for treating retinal degenerations. Tight regulation of transgene expression in Müller cells is likely to be critical to the efficacy of long-term neuroprotective therapies, due to the genetic heterogeneity and progressive nature of retinal disease. To this end, we developed a modified lentiviral (LV) transfer vector (pFTMGW) to accelerate the testing and evaluation of novel transcriptional regulatory elements. This vector facilitates identification and characterization of regulatory elements in terms of size, cell specificity and ability to control transgene expression levels.
  
  
  METHODS
  A synthetic multiple cloning site (MCS) which can accept up to five directionally cloned DNA regulatory elements was inserted immediately upstream of an enhanced green fluorescent protein (eGFP) reporter. A cytomegalovirus (CMV) promoter, required for tat-independent viral packaging, is located around 2 kb upstream of the eGFP reporter and is capable of directing transgene expression. A synthetic transcription blocker (TB) was inserted to insulate the MCS/eGFP from the CMV promoter. We evaluated eGFP expression from pFTMGW and control constructs using flow cytometry and quantitative reverse transcriptase polymerase chain reaction (RT-PCR). We also tested and compared the activity and cell specificity of a computationally identified promoter fragment from the rat vimentin gene (Vim409) in transfection and lentiviral infection experiments using fluorescence microscopy.
  
  
  RESULTS
  Transfection data, quantitative RT-PCR, and flow cytometry show that around 85% of expression from the CMV promoter was blocked by the TB element, allowing direct evaluation of expression from the Vim409 candidate promoter cloned into the MCS. Lentiviruses generated from this construct containing the Vim409 promoter (without the TB element) drove robust eGFP expression in Müller cells in vitro and in vivo.
  
  
  CONCLUSIONS
  The TB element efficiently prevented eGFP expression by the upstream CMV promoter and the novel MCS facilitated testing of an evolutionarily conserved regulatory element. Additional sites allow for combinatorial testing of additional promoter, enhancer, and/or repressor elements in various configurations. This modified LV transfer vector is an effective tool for expediting functional analysis of gene regulatory elements in Müller glia, and should prove useful for promoter analyses in other cell types and tissues.
authors.affiliation: Helen Wills Neuroscience Institute, University of California, Berkeley, CA 94720-3190, USA.
authors.forename: Scott F
authors.initials: SF
authors.lastname: Geller
authors.forename: Phillip S
authors.initials: PS
authors.lastname: Ge
authors.forename: Meike
authors.initials: M
authors.lastname: Visel
authors.forename: Kenneth P
authors.initials: KP
authors.lastname: Greenberg
authors.forename: John G
authors.initials: JG
authors.lastname: Flannery
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods; D003587:Cytomegalovirus / Q000235:genetics; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D016086:Lentivirus; D009457:Neuroglia / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012160:Retina / Q000166:cytology / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection; D019076:Transgenes; D014746:Vimentin / Q000235:genetics
chemical_list: D005456:Fluorescent Dyes; D014746:Vimentin; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins