title: Delivery and long-term expression of a 135 kb LDLR genomic DNA locus in vivo by hydrodynamic tail vein injection.
abstract: BACKGROUND
  The delivery of a complete genomic DNA locus in vivo may prove advantageous for complementation gene therapy, especially when physiological regulation of gene expression is desirable. Hydrodynamic tail vein injection has been shown to be a highly efficient means of non-viral delivery of plasmid DNA to the liver. Here, we apply hydrodynamic tail vein injection to deliver and express large genomic DNA inserts > 100 kb in vivo.
  
  
  METHODS
  Firstly, a size series (12-172 kb) of bacterial artificial chromosome (BAC) plasmids, carrying human genomic DNA inserts, episomal retention elements, and the enhanced green fluorescent protein (EGFP) reporter gene, was delivered to mice by hydrodynamic tail vein injection. Secondly, an episomal BAC vector carrying the whole genomic DNA locus of the human low-density lipoprotein receptor (LDLR) gene, and an expression cassette for the LacZ reporter gene, was delivered by the same method.
  
  
  RESULTS
  We show that the efficiency of delivery is independent of vector size, when an equal number of plasmid molecules are used. We also show, by LacZ reporter gene analysis, that BAC delivery within the liver is widespread. Finally, BAC-end PCR, RT-PCR and immunohistochemistry demonstrate plasmid retention and long-term expression (4 months) of human LDLR in transfected hepatocytes.
  
  
  CONCLUSIONS
  This is the first demonstration of somatic delivery and long-term expression of a genomic DNA transgene > 100 kb in vivo and shows that hydrodynamic tail vein injection can be used to deliver and express large genomic DNA transgenes in the liver.
authors.affiliation: Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, UK.
authors.forename: Olivia C
authors.initials: OC
authors.lastname: Hibbitt
authors.forename: Richard P
authors.initials: RP
authors.lastname: Harbottle
authors.forename: Simon N
authors.initials: SN
authors.lastname: Waddington
authors.forename: Christine A
authors.initials: CA
authors.lastname: Bursill
authors.forename: Charles
authors.initials: C
authors.lastname: Coutelle
authors.forename: Keith M
authors.initials: KM
authors.lastname: Channon
authors.forename: Richard
authors.initials: R
authors.lastname: Wade-Martins
mesh_terms: D000818:Animals; D020029:Base Pairing / Q000235:genetics*; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D004247:DNA / Q000378:metabolism*; D005260:Female; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D015894:Genome, Human / Q000235:genetics*; D006801:Humans; D007267:Injections; D008099:Liver / Q000166:cytology / Q000378:metabolism; D051379:Mice; D010957:Plasmids / Q000378:metabolism; D011973:Receptors, LDL / Q000235:genetics*; D013997:Time Factors; D019076:Transgenes; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D011973:Receptors, LDL; D004247:DNA; D001616:beta-Galactosidase