Abstract
Extrachromosomal gene expression vectors that contain native genomic gene expression elements have numerous advantages over traditional integrating mini-gene vectors. In this protocol chapter we describe our work using episomal vectors where expression of a cDNA is controlled by a 10 kB piece of genomic DNA encompassing the promoter of the low density lipoprotein receptor. We explain methods to sub-clone large genomic inserts into gene expression vectors. We also illustrate various methods employed to ascertain whether expression from these vectors is robust and physiologically relevant by investigating their sensitivity to changes in cellular milieu. Delivery of gene expression vectors in vivo is also described using hydrodynamic tail vein injection, a high pressure, high volume tail vein injection used for liver-directed gene transfer.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Hibbitt OC, Wade-Martins R. (2006) Delivery of large genomic DNA inserts >100 kb using HSV-1 amplicons. Curr Gene Ther;6(3):325–36.
Lufino MM, Edser PA, Wade-Martins R. (2008) Advances in high-capacity extrachromosomal vector technology: episomal maintenance, vector delivery, and transgene expression. Mol Ther;16(9):1525–38.
Hacein-Bey-Abina S, von Kalle C, Schmidt M, et al. (2003) A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med;348(3):255–6.
Bushman F, Lewinski M, Ciuffi A, et al. (2005) Genome-wide analysis of retroviral DNA integration. Nat Rev Microbiol;3(11):848–58.
Wade-Martins R, Saeki Y, Chiocca EA. (2003) Infectious delivery of a 135-kb LDLR genomic locus leads to regulated complementation of low-density lipoprotein receptor deficiency in human cells. Mol Ther;7(5 Pt 1):604–12.
Hibbitt OC, Harbottle RP, Waddington SN, et al. (2007) Delivery and long-term expression of a 135 kb LDLR genomic DNA locus in vivo by hydrodynamic tail vein injection. J Gene Med;9(6):488–97.
Lufino MM, Manservigi R, Wade-Martins R. (2007) An S/MAR-based infectious episomal genomic DNA expression vector provides long-term regulated functional complementation of LDLR deficiency. Nucleic Acids Res;35(15):e98.
Hibbitt OC, McNeil E, Lufino MM, Seymour L, Channon K, Wade-Martins R. (2010) Long-term Physiologically Regulated Expression of the Low-density Lipoprotein Receptor In Vivo Using Genomic DNA Mini-gene Constructs. Mol Ther 18(2):317–26. Epub 2009 Oct 27.
Goldstein JL, Kita T, Brown MS. (1983) Defective lipoprotein receptors and atherosclerosis. Lessons from an animal counterpart of familial hypercholesterolemia. N Engl J Med;309(5):288–96.
Tolleshaug H, Hobgood KK, Brown MS, Goldstein JL. (1983) The LDL receptor locus in familial hypercholesterolemia: multiple mutations disrupt transport and processing of a membrane receptor. Cell;32(3):941–51.
Cichon G, Willnow T, Herwig S, et al. (2004) Non-physiological overexpression of the low density lipoprotein receptor (LDLr) gene in the liver induces pathological intracellular lipid and cholesterol storage. J Gene Med;6(2):166–75.
Heeren J, Steinwaerder DS, Schnieders F, Cichon G, Strauss M, Beisiegel U. (1999) Nonphysiological overexpression of low-density lipoprotein receptors causes pathological intracellular lipid accumulation and the formation of cholesterol and cholesteryl ester crystals in vitro. J Mol Med;77(10):735–43.
Zhang G, Gao X, Song YK, et al. (2004) Hydroporation as the mechanism of hydrodynamic delivery. Gene Ther;11(8):675–82.
Zhang G, Song YK, Liu D. (2000) Long-term expression of human alpha1-antitrypsin gene in mouse liver achieved by intravenous administration of plasmid DNA using a hydrodynamics-based procedure. Gene Ther;7(15):1344–9.
Zhang X, Dong X, Sawyer GJ, Collins L, Fabre JW. (2004) Regional hydrodynamic gene delivery to the rat liver with physiological volumes of DNA solution. J Gene Med;6(6):693–703.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Hibbitt, O., Wade-Martins, R. (2011). High Capacity Extrachromosomal Gene Expression Vectors. In: Hadlaczky, G. (eds) Mammalian Chromosome Engineering. Methods in Molecular Biology, vol 738. Humana Press. https://doi.org/10.1007/978-1-61779-099-7_2
Download citation
DOI: https://doi.org/10.1007/978-1-61779-099-7_2
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-098-0
Online ISBN: 978-1-61779-099-7
eBook Packages: Springer Protocols