Lentiviral Vectors in Huntington’s Disease Research and Therapy - Viral Vector Approaches in Neurobiology and Brain Diseases - page 193

A A

Δ R U3 psi RRE cPPT Promoter Transgene WPRE miRT Δ R U5

SIN

H C E E

S N S'

Fig. 3 Schematic diagram of an SIN transfer plasmid. Dark gray elements represent the expression cassette;
the bright gray element is optional and its presence depends on biological need. psi, HIV-1 packaging signal;
RRE, rev-responsive element; cPPT, central polypurine tract; WPRE, woodchuck posttranscriptional regulatory
element; miRT, miRNA-target sequence; A, Asp718 ; C, Cla I; E, Eco RI; H, Hin dIII; N, Not I; S, Sac I; S ′ , Sal I

oligodendrocytes [ 65 , 66 ] to characterize functional elements in the
human genome (ENCODE project, for encyclopedia of DNA ele-
ments) and to develop mini-promoters (Pleiade Promoter Project;
[ 67 - 69 ]). A comprehensive analysis of the transcriptional promoter
structure of 1 % of the human genome (400 promoters) has revealed
a strong correlation between promoter activity and the correspond-
ing endogenous transcript levels, providing the fi rst experimental,
quantitative estimate of the contribution of the promoter to gene
regulation [ 70 - 72 ]. These projects should provide new regulatory
sequences to drive gene expression in a region-specifi c manner in
the brain. In the meantime, a limited number of neuronal promot-
ers [ 73 - 76 ] and glial promoters [ 77 - 80 ] for limiting transgene
expression in the brain are currently available.
Promoters could be inserted into transfer plasmids by classical
molecular biology methods or with Gateway ® technology. We
describe the principal cloning steps here. For further details, please
refer to specialized laboratory manuals [ 81 ].

This approach makes use of unique restriction sites at the 5

ends of each element to facilitate cloning (Fig. 3 ). The LV transfer
plasmid (10

′ and 3 ′

3.4.1 Molecular Cloning
of the Promoter in a
Lentiviral Transfer Plasmid

g) and the plasmid containing the promoter (the
insert plasmid) are digested with the same restriction enzymes
(REs), in a fi nal volume of 50

μ

l, for 2 h (the buffer and tempera-
ture depend on the REs used). If a compatible double digestion is
not possible, an additional blunting step may be required (with the
T4 DNA polymerase, for example). The corresponding DNA frag-
ments are separated by electrophoresis in an agarose gel and puri-
fi ed on commercially available columns. The destination plasmid is
dephosphorylated to prevent self-ligation ( see Note 3 ). Ligation is
performed with T4 DNA ligase by incubation for 1 h at room tem-
perature or for 24 h at 14 °C for blunt-ended fragments. Competent
Escherichia coli cells (DH10B electrocompetent, or TOP10 chemi-
cally competent strains) are transformed the next day, with 2

μ

μ l of

ligation mixture. The bacteria are resuspended in 450 μ l of SOC

medium, spread on selective plates (250 l/plate) and incubated

overnight at 37 °C. Finally, clones are analyzed and insertion of the
transgene is validated by a control RE digestion and sequencing.

μ