Lentiviral Vectors in Huntington’s Disease Research and Therapy - Viral Vector Approaches in Neurobiology and Brain Diseases - page 194

Entry plasmid
x LR Clonase

attL1 Promoter attL2

Conversion cassette

Destination lentiviral plasmid

Cm R

LTR attR1 ccdB attR2 Transgene WPRE LTR

Recombination sites

Lentiviral transfer plasmid

LTR attB1 Promoter attB2 Transgene WPRE LTR

Fig. 4 Gateway ® technology for cloning in an LV transfer plasmid. The promoter is inserted into an entry plas-
mid containing the attL1 and attL2 recombination sites. LV destination vectors are then produced by integrat-
ing a conversion cassette (attR1-ccdB-Cm R -attR2) upstream from the transgene. The LR reaction is then
performed

Gateway ® cloning technology facilitates the insertion of large DNA
fragments into lentiviral transfer plasmids (>7-10 kb). The
Gateway ® system is based on homologous recombination between
the attL and attR sites from the bacteriophage

3.4.2 Cloning of
Promoters with Gateway ®
Technology in Lentiviral
Transfer Plasmids

. A two-step pro-
cedure is used (1) insertion of the promoter of interest into an
entry plasmid containing attL sites and (2) production of a desti-
nation LV transfer plasmid containing a conversion cassette, to
integrate the promoter upstream from the gene of interest. This
conversion cassette contains attR sites, a suicide gene (ccdB, see
Note 4 ) and the chloramphenicol resistance gene for screening of
the clones (Fig. 4 ).
Entry plasmids containing multiple cloning sites (MCS) (for
example, pENTR4 (Invitrogen)) or plasmids suitable for PCR
products (e.g., plasmids predigested to allow oriented cloning such
as example, pENTR-D-TOPO (Invitrogen); see Sect. 3.4.3 ) should
be used for the generation of entry plasmids with promoters.

λ

The choice of the plasmid depends on the polymerase used for
PCR. pENTR-D-TOPO ® is designed for the cloning of blunt-
ended DNA fragments compatible with the Pfx or PfuUltra II
enzymes ( see Note 5 ), whereas pCR2.1-TOPO-TA ® has 3

3.4.3 Insertion of PCR
Products into Entry
Plasmids

-T over-
hangs for the direct ligation of Taq -amplifi ed PCR products.

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3.5 miRNA
Detargeting Strategy

Naldini and coworkers integrate microRNA (miRNA) regulatory
sequences in LVs to restrict transgene expression [ 82 , 83 ]. This
approach is based on the insertion, at the 3

of the transgene, of
copies of a microRNA (miR) target sequence (miRT) specifi c for a
lineage. In a proof-of-principle study for the CNS, we showed that
the integration of the miR124-target (miR124T) sequence into
LVs inhibits transgene expression in neurons and, together with
MOK-G pseudotyping, provides an effective strategy for the selec-
tive expression of transgenes in astrocytes. The brain is enriched in
miR124, which is selectively expressed in neurons and not in astro-
cytes [ 84 - 86 ]. The integration of the miR124T element leads to
posttranscriptional transgene silencing in neurons but not in astro-
cytes (Fig. 5 ). The choice of miRT sequence depends on several

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