Herpes Simplex Virus 1 (HSV-1)-Based Vectors - Viral Vector Approaches in Neurobiology and Brain Diseases - page 57

9 Construction of Recombinant HSV-1 Vectors by Homologous

Recombination in Bacteria

The cloning of large DNA virus genomes, such as that of HSV-1
[ 25 , 26 ] as BACs, has facilitated the easy construction of recombi-
nant viruses by homologous recombination in Escherichia coli . The
protocol generally used is the construction of recombinant HSV-1
by using the λ prophage homologous recombination system (red α

and red genes) and galK selection. The galK selection method is

a two-step system: in the fi rst step, a galK cassette, fl anked by at
least 50 nucleotides of homology to specifi ed positions on the
HSV-1 BAC DNA, is inserted via homologous recombination into
the virus genome ( galK positive selection). In the second step, the
galK cassette is replaced by homologous recombination with an
oligonucleotide or PCR product that contains the transgene of
interest surrounded by appropriate homology arms and selection
against galK . This method allows constructing a recombinant
HSV-1 within 2-3 weeks.

β

10 Construction of HSV-1-Based Amplicon Vectors

HSV-1-based amplicon vectors carry no viral genes; they are there-
fore replication defective and depend on helper functions for pro-
duction. Helper functions can be provided either by replication
competent, but packaging-defective HSV-1 genomes cloned as set
of cosmids [ 27 ] or BAC [ 28 ]. Following transfection into mam-
malian cells, sets of cosmids that overlap and represent the entire
HSV-1 genome can form circular replication-competent viral
genomes via homologous recombination. These reconstituted viral
genomes give rise to infectious virus progeny. Similarly, BACs that
contain the entire HSV-1 genome also produce infectious virus
progeny in transfected cells. If the viral DNA packaging/cleavage
( a ) signals are deleted from the HSV-1 cosmids or HSV-1 BAC,
reconstituted virus genomes are packaging defective; however,
even in the absence of the a signals, these genomes can still provide
all helper functions required for the replication and packaging of
cotransfected amplicon DNA. The resulting amplicon vector stocks
are essentially free of helper virus contamination.
Alternatively, helper-free amplicon vector stocks can be prepared
using a helper system based on the deletion of the a signals of the
helper virus genome by Cre/loxP-based site-specifi c recombination,
in order to inhibit its cleavage/encapsidation in the cells that are pro-
ducing the amplicons [ 29 ]. This helper virus, named HSV-1-LaL J

helper, carries a unique and ectopic a signal, fl anked by two loxP sites
in parallel orientation. This is therefore a Cre-sensitive virus that can
express all virus proteins but cannot be packaged in Cre-expressing

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