Lentiviral Vectors as Research Tools in Neurobiology: Design and Production - Viral Vector Approaches in Neurobiology and Brain Diseases - page 4

2.2 Targeting the
Gene Expression

In the vast majority of gene-delivery applications, a precise targeting
of gene expression is mandatory. To achieve this results there are
two steps of the LVs transduction process that can be modified:
vector entry in the host cell and gene expression.
Pseudotyping is defined as the combination of a viral particle
with the glycoprotein of a different virus. As stated in the introduc-
tion, LVs are commonly pseudotyped with VSV-G, which guarantees
a broad-range tropism. However, this versatility, which is often an
advantage in research, could be a serious limitation for further
clinical use of the vectors. For this reason in many studies, pseudo-
typing with other virus glycoproteins is often used. A comprehen-
sive list of all the possible options is beyond the scope of this
chapter, the interested reader can find an exhaustive review in the
work of Cronin and colleagues [ 23 ].
LVs can also allow a tight spatio-temporal control transgene
expression. There are two main possibilities: the use of tissue-
specific promoters or the use of inducible vector systems. If the
promoter that drives the expression is tissue specific, then the
expression can be restricted to a specific cell type. Virtually every
kind of small- and medium-size promoter can be cloned in a LV.
For example to restrict to neurons the expression of some trans-
genes we successfully used [ 24 ] the −238/+25 region of the pro-
moter of the α isoform of the mouse Ca 2+ /calmodulin-dependent
protein kinase II (αCaMKII) gene [ 25 , 26 ]. This short version of
the αCaMKII promoter still retains its neuronal specificity but is
deprived of the −199/−275 strong silencer element upstream of
the transcriptional starting site. Another possibility is to allow an
inducible expression in LV with the TET system. Originally devel-
oped in the Bujard's laboratory [ 27 ], this system is based on the
tetracycline-resistance operon of E. coli and relies on two compo-
nents: the tetracycline-controlled transactivator (tTA) and the tet-
racycline-response element (TRE). Initially developed as a Tet-Off
system only (in the bacterial operon the tTA transactivator binds to
TRE and initiate transcription only in absence of tetracycline), a
screening of random mutations in the tTA led to design an alter-
native Tet-On system: this mutant tTA, referred as reverse tTA
(rtTA), is active only in the presence of tetracycline or the brain-
blood barrier permeable analogue doxycycline [ 28 ]. Among oth-
ers, the rtTA-M2 version has been shown to be the most efficient
in a LV configuration [ 29 ]. To date, various inducible LV systems
are commercially available and recently an all-in-one LV system
based on the original bacterial Tet repressor (less toxic then the
derivative tTA) has been developed [ 30 ].

2.3 Gene Silencing
Vectors

Gene silencing vectors have become an essential technology to
study gene functions. These vectors use RNA interference (RNAi)
mediated gene silencing and can be divided in two categories:
short hairpin RNAs (shRNAs) and microRNAs (miRNAs). Both