Site directed mutagenesis

• Classically, mutants are generated by treating

the test organism with chemical or physical
agents that modify DNA (mutagens).

• Instead of crudely mutagenizing many cells or

organisms and then analyzing many thousands
or millions of offspring to isolate a desired
mutant, it is now possible to change
specifically any given base in a cloned DNA
sequence. This technique is known as site-
directed mutagenesis.
Oligonucleotide-directed mutagenesis
• All oligonucleotide-directed mutagenesis is
based on the same concept - an oligonucleotide
encoding the desired mutation(s) is annealed to
one strand of the DNA of interest and serves as
a primer for initiation of DNA synthesis. In this
manner, the mutagenic oligonucleotide is
incorporated into the newly synthesized strand.

• Mutagenic oligonucleotides incorporate at least

one base change but can be designed to
generate multiple substitutions, insertions or
deletions.
 Primer extension method
• Primer extension (the single-primer method) is a simple
method for site-directed mutation.

• The method involves in vitro DNA synthesis with a

chemically synthesized oligonucleotide (7–20
nucleotides long) that carries a base mismatch with the
complementary sequence.

• The method requires that the DNA to be mutated is

available in single-stranded form, and cloning the gene in
M13-based vectors makes this possible.
C
 Primer extension method
• The synthetic oligonucleotide primes DNA
synthesis and is itself incorporated into the
resulting heteroduplex molecule.

• After transformation of the host E. coli, this

heteroduplex gives rise to homoduplexes whose
sequences are either that of the original wild-
type DNA or that containing the mutated base.

• The mutant clones can be screened by nucleic

acid hybridization or sequencing.
 Primer extension method
• A variation of the procedure involves
oligonucleotides containing inserted or
deleted sequences. As long as stable
hybrids are formed with single-stranded
wild-type DNA, priming of in vitro DNA
synthesis can occur, leading to mutation.
 Single-primer method
• The single-primer method has a number of
deficiencies;

• The double-stranded heteroduplex molecules

that are generated will be contaminated both by
any single-stranded non-mutant template DNA
and by partially double-stranded molecules.

• The presence of these species considerably

reduces the proportion of mutant progeny.
 Problems of single-primer method
• Following transformation and in vivo DNA synthesis,
segregation of the two strands of the heteroduplex
molecule can occur, yielding a mixed population of
mutant and non-mutant progeny.

• Mutant progeny have to be purified away from parental

molecules, and this process is complicated by the cell’s
mismatch repair system (in vitro-generated mutant
strand is repaired by the cell so that the majority of
progeny are wild type).

• Another disadvantage of all of the primer extension

methods is that they require a single-stranded template.
 Site-directed mutagenesis by PCR

• In contrast, PCR-based mutagenesis uses

single-stranded or double-stranded,
circular or linear DNA.

• In comparison with single-stranded DNAs,

double-stranded DNAs are much easier to
prepare. Also, inserts are in general more
stable with double-stranded DNAs.
 Site-directed mutagenesis by antibiotic selection

• The basis for site-directed mutagenesis by positive

antibiotic selection is that a selection oligonucleotide or
oligonucleotides are simultaneously annealed, with the
mutagenic oligonucleotide, to repair an antibiotic
resistance gene

• Selection for the mutant strand is enabled by antibiotic

resistance of the mutated DNA and sensitivity of the non-
mutated strand.

• This approach offers a very efficient means to generate

an indefinite number of the desired mutations with little
hands-on time.
 Cassette mutagenesis
• For mutating more than one codon, requirement
for different mutagenic oligonucleotides (in PCR-
based mutagenesis) is a problem, assuming
only one codon will be mutated by one
oligonucleotides .

• The alternative is cassette mutagenesis, which

involves replacing a fragment of the gene with a
different fragments containing the desired codon
changes.
Oligonucleotides can also encode a library of mutations by
randomizing the base composition at sites during chemical
synthesis resulting in degenerate or "doped" oligonucleotides.
(Doped oligonucleotides usually refers to the use of unequal
amounts of each of the four standard dNTPs in oligonucleotide
synthesis.)
 Site-directed mutagenesis by the use of a unique restriction site

• In this approach, a selection oligonucleotide containing a

mutated sequence for a unique restriction site is annealed
simultaneously with the mutagenic oligonucleotide.

• The selection oligonucleotide renders the nonessential site

immune to restriction by the corresponding enzyme.

• Selection for the mutant strand is enhanced by digesting the

resulting pool of plasmids with the unique restriction enzyme.

• The digestion linearizes the parental plasmid thereby

effectively decreasing its ability to transform bacteria. This
approach requires that a unique restriction site be available,
and that the subsequent digestion be optimal.
 Applications of site directed mutagenesis

• Almost every property of a protein including

solubility, thermostability, specificity, catalysis,
binding etc can be altered by site directed
mutagenesis.
• E.g. rate of catalysis, substrate, specificity, pH-
rate profile, and stability to oxidative, thermal,
and alkaline inactivation has been altered for
serine proteases.
• E.g. a number of variants of subtilisin have been
created with greatly enhanced solvent stability

• Similarly, gene expression modules

(promoter, enhancer etc) or sequences
affecting RNA stability can be changed.
Cloning and expression in Bacteria
 Expression Stategies for heterologous genes

• Expression vectors are required if one wants to

prepare RNA probes from the cloned gene or protein.

• A comparison of prokaryotic promoters has led to the

formulation of a consensus sequence (−35 region 5′-
TTGACA-) and (−10 region, or Pribnow box, 5′-TATAAT).

• Other bases flanking these regions can also affect

promoter activity.

• The distance between the −35 and −10 regions is also

important.
 Expression Stategies

• Bacterial DNA has two types of transcription termination

site: factor-independent and factor-dependent.

• The factor-independent transcription terminators have

similar sequences: an inverted repeat followed by a
string of A residues.

• Most expression vectors incorporate a factor-

independent termination sequence downstream from
the site of insertion of the cloned gene.
 Expression Stategies

• When maximizing gene expression it is not

enough to select the strongest promoter
possible: the toxic effects of
overexpression on the host cell also
need to be considered.
 λ PL promoter system
• The λ PL promoter system λ PL promoter
system combines very tight transcriptional
control with high levels of gene expression.

• This is achieved by putting the cloned gene

under the control of the PL promoter carried on a
vector, while the PL promoter is controlled by a cI
repressor gene in the E. coli host.
 Rate of translation
• Although many factors can influence the rate of
translation, the most important is the interaction of the
ribosome with the bases immediately upstream from the
initiation codon of the gene.

• In bacteria, a key sequence is the ribosome-binding site

or Shine–Dalgarno (S–D) sequence. The degree of
complementarity of this sequence with the 16S rRNA can
affect the rate of translation.

• The spacing between the S-D sequence and the

initiation codon is also important.
 Purification tags
• Examples of tags include glutathione-S-transferase
(binds glutathione), the MalE (maltose-binding protein),
and multiple histidine residues (binds nickel columns),
which can easily be purified by affinity
chromatography.

• Tag vectors are usually constructed so that the coding

sequence for an amino acid sequence cleaved by a
specific protease is inserted between the coding
sequence for the tag and the gene being expressed.

• After purification, the tag protein can be cleaved off with

the specific protease to leave a normal or nearly normal
protein.
 Purification tags
• It is also possible to include in the tag a protein
sequence (e.g. short antibody recognition sequences)
that can be assayed easily.

• Polyhistidine tag can function for both assay and

purification.

• The affinity purification systems described here suffer

from the disadvantage that a protease is required to
separate the target protein from the affinity tag.

• Also, the protease has to be separated from the

protein of interest.
Purification tags
 Self-cleaving purification tags
• protein splicing element, an intein, undergoes a
self-cleavage reaction at its N terminus at low
temperatures in the presence of thiols, such as cysteine,
dithiothreitol, or β-mercaptoethanol.

• DNA encoding a chitin-binding domain Is added to the

C terminus of the intein for affinity purification

• After purification on chitin columns, the tag can be self-

cleaved.
 Minimizing inclusion-body formation

• Vectors are available that promote solubilization

of expressed proteins.

• There are a number of reports which show that

lowering the temperature of growth increases
the yield of correctly folded, soluble protein.

• Media compositions and pH values that reduce

the growth rate also reduce inclusion-body
formation.
 Genetic methods of preventing inclusion-body formation

• Host cell can be engineered to overproduce a

chaperon.

• The second method involves making minor changes to

the amino acid sequence of the target protein.

• Many proteins produced as insoluble aggregates in

their native state are synthesized in soluble form as
thioredoxin fusion proteins.

• Many of the vectors designed for high-level expression

also contain translation-initiation signals optimized for E.
coli expression.
 Genetic methods of preventing inclusion-body formation

• Alternative way of keeping recombinant

proteins soluble is to export them to the
periplasmic space.

• Proteins that are synthesized with signal

sequences are exported from the cell.

• It is possible to engineer proteins such that

they are transported through the outer
membrane and are secreted into the growth
medium.
 pLITMUS vectors
• The vectors are small (<3 kb) and high copy number.

• Are used for the generation of RNA probes.

• The polylinkers are located in the lacZ′ gene and inserts in the
polylinker prevent α-complementation. Thus blue/white
screening (see can be used to distinguish clones with inserts
from those containing vector only.

• The LITMUS polylinkers contain a total of 32 unique

restriction sites. Twenty-nine of these enzymes leave four-
base overhangs and three leave blunt ends. The three blunt
cutting enzymes have been placed at either end of the
polylinker and in the middle of it.
 pLITMUS vectors
• The vectors carry both the pUC and the M13 ori regions. Under
normal conditions the vector replicates as a double-stranded plasmid
but, on infection with helper phage (M13KO7), single-stranded
molecules are produced and packaged in phage protein.

• The polylinker regions are flanked by two modified T7RNA

polymerase promoters. Each contains a unique restriction site (SpeI or
AflII) that has been engineered into the T7 promoter consensus
sequence such that cleavage with the corresponding endonuclease
inactivates that promoter.

• Both promoters are active despite the presence of engineered sites.

• The single-stranded molecules produced on helper phage addition

have all the features necessary for DNA sequencing.
.
 pBAD vectors
• Offer extremely tight control of expression of cloned genes.

• pBAD vectors carry the promoter of the araBAD (arabinose) operon

and the gene encoding the positive and negative regulator of
this promoter, araC. AraC is a transcriptional regulator that
forms a complex with l-arabinose.

• In the absence of arabinose, AraC binds to the O2 and I1 half-sites

of the araBAD operon, forming a 210 bp DNA loop and
thereby blocking transcription.

• As arabinose is added to cAMP synthesis, thereby decreasing

expression from the araBAD promoter. Thus one can titrate the level
of cloned gene product by varying the glucose and arabinose
content of the growth medium.
 pBAD vectors
• Three different variants of vrctor (A, B, C) allow the insert
to be placed in each of the three translational reading
frames (each with a polylinker in a different reading
frame).

• The cleavage site and polyhistidines can be placed at

the C terminus.

• If the cloned gene product itself contains an

enterokinase cleavage site, then an alternative protease,
such as thrombin or factor Xa, with a different cleavage
site can be used.
 Codon optimization
• The codon composition following the AUG start
codon affect the rate of translation. For example,
a change in the third position of the fourth codon
of a human -interferon gene resulted in a 30-
fold change in the level of expression.

• The composition of the triplet immediately

preceding the AUG start codon also affects the
efficiency of translation.
 Codon optimization
• Also, there is a strong bias in the second codon of many
natural mRNAs, which is quite different from the general
bias in codon usage.

• Highly expressed genes have AAA (Lys) or GCU (Ala) as

the second codon.

• DevIin et al. (1988) changed all the G and C nucleotides

for the first four codons of granulocyte colony-stimulating
factor gene and expression increased from undetectable
to 17% of total cell protein.
 Codon optimization
• In the E. coli rnd gene there is a run of eight uracil residues.
Changing two to five of these residues has no effect on
mRNA levels but reduces translation by up to 95%.

• Etchegaray and Inouye (1999) have identified an element

downstream of the initiation codon, the downstream box,
which facilitates formation of the translation-initiation
complex.

• The sequence of the 3′ untranslated region of the mRNA

can also be important. If this region is complementary to
sequences within the gene, hairpin loops can form and
hinder ribosome movement along the messenger.
 Codon optimization
• The genetic code is degenerate, and hence for most of the amino
acids there is more than one codon. However, in all genes, whatever
their origin, the selection of synonymous codons is distinctly non-
random.

• The bias in codon usage has two components: correlation with tRNA
availability in the cell, and non-random choices between pyrimidine-
ending codons.

• Ikemura (1981a) measured the relative abundance of the 26 known

tRNAs of E. coli and found a strong positive correlation between tRNA
abundance and codon choice.

• Later, Ikemura (1981b) noted that the most highly expressed genes in
E. coli contain mostly those codons corresponding to major tRNAs
but few codons of minor tRNAs.
•
 Codon optimization
• In contrast, genes that are expressed less well use more
suboptimal codons.

• Forman et al. (1998) noted significant misincorporation

of lysine, in place of arginine, when the rare AGA codon
was included in a gene overexpressed in E. coli.

• It should be noted that the bias in codon usage even

extends to the stop codons. UAA is favored in genes
expressed at high levels, whereas UAG and UGA are
used more frequently in genes expressed at a lower
level.
 Beyond codon optimization
• . The ability to incorporate unnatural amino acids into
proteins in vivo would permit the production of large
quantities of proteins with novel properties. For example,
the replacement of methionine with selenomethionine
facilitates the determination of the three-dimensional
structure of proteins.

• While it is possible to “force” bacteria to incorporate

unnatural amino acids into proteins, a better method is to
engineer the translational apparatus. This is achieved by
generating an aminoacyl-tRNA synthetase and tRNA pair
that function independently.
 Cloning and expression in Yeast
• Fungi offer a number of advantages, such as
the ability to glycosylate protein, the absence of
pyrogenic toxins, and in the case of the
methylotrophic yeast Pichia pastoris, the ability
to get very high yields of recombinant proteins.

• Yeasts and other fungi are widely used in the

production of food and beverages and
recombinant DNA technology can be used to
enhance their desirable properties.
 Cloning and expression in Yeast
• Like E. coli, fungi are not naturally transformable and
artificial means have to be used for introducing foreign
DNA.

• One method involves the use of spheroplasts (i.e. wall-

less cells). In this method, the cell wall is removed
enzymically and the resulting spheroplasts are fused with
ethylene glycol in the presence of DNA and CaCl2.

• The spheroplasts are then allowed to generate new cell

walls in a stabilizing medium containing 3% agar.
 Cloning and expression in Yeast
• Electroporation provides a simpler and more
convenient alternative to the use of spheroplasts. Cells
transformed by electroporation can be selected on the
surface of solid media.

• DNA can also be introduced into yeasts and filaentous

fungi by conjugation. plasmids, such as R751 (IncPβ)
and FncF), could facilitate plasmid transfer from E. coli to
S. cerevisiae and Schizosaccharomyces pombe. Ti
plasmid, and part of this plasmid (the transferred DNA
(T-DNA) can be conjugally transferred to protoplasts of
S. cerevisiae.
 Cloning and expression in Yeast
• Four types of plasmid vector have been developed for
yeasts: yeast episomal plasmids (YEps), yeast
replicating plasmids (YRps), yeast centromere plasmids
(YCps), and yeast artificial chromosomes (YACs).

• All of them have features in common. First, they all

contain unique target sites for a number of restriction
endonucleases. Secondly, they can all replicate in E.
coli, often at high copy number. Finally, they all
employ markers that can be selected readily in yeast and
which will often complement the corresponding
mutations in E. coli as well.
Cloning and expression in Yeast
• The four most widely used markers are His3,
Leu2, Trp1, and Ura3. Mutations in the cognate
chromosomal markers are recessive, and non-
reverting mutants are available.

• Two yeast selectable markers, Ura3 and Lys2,

have the advantage of offering both positive and
negative selection. Positive selection is for
complementation of auxotrophy. Negative
selection is for ability to grow on medium
containing a compound that inhibits the growth
of cells expressing the wild-type function.
 Yeast promoters
• Yeast promoters are more complex than
bacterial promoters.

• Several consensus sequences are found at the

transcription-initiation site.

• Other than 1st (TC(G/A)A and PuPuPyPuPu),

and 2nd (TATA box), the third and fourth
structural elements are upstream activating
sequences (UASs) and upstream repressing
sequences (URSs).
 Cell surface display
• Heterologous proteins can be synthesized
as fusions for display on the cell surface of
yeast.

• Yeast surface display makes use of the

cell surface receptor α-agglutinin (Aga).
 Baculovirus based systems
• Baculovirus vectors promote high-level
transgene expression in insect cells, but can
also infect mammalian cells (without producing
progeny virions).

• Baculoviruses have rod-shaped capsids and

large, double-stranded DNA genomes.

• One group of baculoviruses, known as the

nuclear polyhedrosis viruses, produces nuclear
occlusion bodies.
 Baculovirus based systems
• The occlusion bodies are made up of polyhedrin, which is
expressed at very high levels.

• The nuclear occlusion stage of the infection cycle is non-

essential for the productive infection of cell lines, thus the
polyhedrin gene can be replaced with foreign DNA, which
can be expressed at high levels under the control of the
polyhedrin promoter.

• Polyhedrin gene replacement vectors are the most

popular due to the high level of recombinant protein that
can be expressed (up to 1 mg per 106 cells)

• Two baculoviruses have been extensively developed as

vectors, namely the Autographa californica multiple
nuclear polyhedrosis virus (AcMNPV) and the Bombyx
mori nuclear polyhedrosis virus (BmNPV).
 Baculovirus based systems
• limitations of baculovirus expression systems:
• Glycosylation pathway in insects differs from that in
mammals. insect cell lines having ability to carry out
mammalian-type post-translational modifications can be
used, or, appropriate glycosylation enzymes can be
expressed along with the transgene.

• Since the baculovirus genome is large (130 kb), cloning

is usually done by homologous recombination. A
problem with homology-based strategies is that
recombinants are generated at a low efficiency (0.5–
5%). The proportion of recombinants can be increased
by using linear derivatives of the wild-type baculovirus
genome containing large deletions, which can be
repaired only by homologous combination with the
targeting vector. This has been resulted in the production
of up to 90% recombinant plaques (see next slide).
 Baculovirus based systems
• Selection:

• OB assay: Occlusion bodies produced by wild-

type viruses cause the microscopic viral plaques
to appear opalescent if viewed under an oblique
light source (OB+), while recombinant plaques
appear clear (OB–)

• Insertion of the E. coli lacZ gene in frame into

the polyhedrin coding region allows blue-white
screening of recombinants in addition to the OB
assay.
 Baculovirus based systems
• Baculoviruses are useful not only for the delivery
of foreign genes into mammalian cells, but also
for the delivery of other viruses.

• Baculoviruses can also be used to improve the

production of recombinant viral vectors.

• Baculovirus vectors have been developed, which

contain E. coli F plasmid (called a bacmid), and
the target site for the transposon Tn7.
Cloning and expression in Mammalian systems

• Mammalian cells are the most widely used

hosts for gene delivery, since they allow the
production of recombinant human proteins with
authentic post-translational modifications
that are not carried out by bacteria, yeast, or
plants.

• Efficient vector systems and transformation

methods for animal cells are based on viral,
bacterial, and synthetic delivery vectors.
Modules for mammalian expression
• Certain viruses contain strong promoters and enhancers that
function in a wide range of cell types, and several of these have
been used in plasmid vectors for mammalian expression.

• Polyadenylation signals (terminators) are required in eukaryotic

genes to generate a defined 3′ end to the mRNA. This poly (A) tail is
required for the export of mRNA into the cytoplasm, and also
increases its stability.

• 5′ and 3′ UTRs, and other codons influence gene expression.

• Since specific types of modification occur in particular cell

compartments, it is necessary to consider strategies for targeting
the recombinant protein to the correct compartment to ensure that it
is appropriately modified. Proteins that need to be glycosylated, for
example, must be targeted to the secretory pathway using a signal
peptide.
 Expression in mammalian system
• Transformation of the animal germline
requires gene transfer to pluripotent or
totipotent cells, such as eggs, early embryos,
isolated germ cells, or gametes.

• Exceptionally, germline transformation can be

achieved by the transformation of cultured
cells derived from pluripotent cell lines, such
as murine embryonic stem (ES) cells, or the
transfer of somatic nuclei into enucleated
eggs.
 Four major strategies for gene
transfer to animal cells

• Transduction: recombinant viral genome,

exploiting the virus’s natural ability to infect
and transfer nucleic acid into animal cells.

• Bactofection: Delivery using bacterial vectors is

a more recent development, which in most
cases relies on bacteria which also invade
animal cells. In this case, however, the
transgene is delivered not as part of the bacterial
genome, but on a plasmid which is carried
by the bacterium.
 Four major strategies for gene
transfer to animal cells
• Non-biological methods (transfection)

• Chemical transfection methods, cells are persuaded to take up DNA

from their surroundings when the DNA is presented as a synthetic
complex – either a complex with an overall positive charge,
allowing it to interact with the negatively charged cell membrane
and promote uptake by endocytosis; or a lipophilic complex
which fuses with the membrane and deposits the transgene
directly into the cytoplasm. The calcium phosphate method
involves the formation of a co-precipitate which is taken up by
endocytosis.

• Physical transfection methods, naked DNA is introduced directly

into the cell by exploiting a physical force. Examples of physical
transfection methods include microinjection, particle
bombardment, ultrasound, and electroporation.
 Gene-transfer
• Whichever gene-transfer strategy is
chosen, the result is transformation, i.e. a
change of the recipient cell’s genotype
caused by the acquired transgene.

• This change in genotype may be

transient or stable, depending on the
type of vector and the fate of the
introduced genetic material.
 Selectable markers for mammalian system
• Endogenous selectable markers are already present in
the cellular genome, and mutant cell lines are required
when they are used.

• Major disadvantage of endogenous markers is that

they can only be used with mutant cell lines in which the
corresponding host gene is non-functional.

• Most genes do not generate a conveniently selectable

phenotype, and the isolation of transformants in such
experiments was problematic.

• Endogenous markers have therefore been largely

superseded by so-called dominant selectable markers.
 Dominant selectable markers
• Confer a phenotype that is entirely novel
to the cell and can hence be used in any
cell type. Such markers are usually drug-
resistance genes of bacterial origin, and
transformed cells are selected on a
medium that contains the drug.
 Reporter genes
• These differ from selectable marker genes in that they
do not confer a property that allows transformed cells to
survive under selective conditions. Instead, they encode
a product that can be detected using a simple and
inexpensive assay.

• When controlled by a strong constitutive promoter,

reporter genes are often used as markers to confirm
transient or stable transformation.

• Importantly, the assays used to detect reporter-gene

activity are quantitative, so they can also be used to
measure transformation efficiency.
 Transient transformation
• The simplest way to achieve the transient transformation of
animal cells is to use a plasmid vector lacking an origin of replication
functional in the host.

• Non-replicating plasmid vectors persist for a short time in an

extrachromosomal state.

• Although the vector cannot replicate, gene expression from a

mammalian transcription unit is possible for as long as the plasmid
remains.

• Used for transient assays of gene expression and the recovery of

moderate amounts of recombinant protein.

• No regime of selection is required, the cells are generally

transfected, assayed after one or two days, and then discarded.
Modules for mammalian expression
• Certain viruses contain strong promoters and enhancers that
function in a wide range of cell types, and several of these have
been used in plasmid vectors for mammalian expression.

• Polyadenylation signals (terminators) are required in eukaryotic

genes to generate a defined 3′ end to the mRNA. This poly (A) tail is
required for the export of mRNA into the cytoplasm, and also
increases its stability.

• 5′ and 3′ UTRs, and other codons influence gene expression.

• Since specific types of modification occur in particular cell

compartments, it is necessary to consider strategies for targeting
the recombinant protein to the correct compartment to ensure that it
is appropriately modified. Proteins that need to be glycosylated, for
example, must be targeted to the secretory pathway using a signal
peptide.
The figure below shows the Invitrogen vector
pSecTag2, which incorporates a sequence
encoding the murine immunoglobulin light-
chain signal peptide for high-efficiency
targeting to the secretory pathway.
 Viral vectors
• Adenoviruses have been widely used as gene-transfer
and expression vectors because they have many
advantageous features, including stability, a high
capacity for foreign DNA, a wide host range that
includes non-dividing cells, and the ability to
produce high-titer stocks (up to 10 11 pfu/ml). They are
suitable for transient expression in dividing cells
because they do not integrate efficiently into the
genome, but prolonged expression can be achieved in
post-mitotic cells such as neurons .

• Baculovirus vectors are used mainly for high-level

transient protein expression in insects and insect cell
lines.
 Viral vectors
• The herpesviruses are large double-stranded DNA viruses that
include Epstein–Barr virus (EBV),

• Unlike EBV, which is used as a replicon vector, HSV-I has been

developed as a transduction vector. Viral replication can occur in
many cell types in a wide range of species if the genome is
introduced by transfection, but HSV vectors are particularly
suitable for gene therapy in the nervous system because the virus
is remarkably neurotropic.

• Retrovirus vectors integrate efficiently into the host-cell genome.

• Retroviruses are RNA viruses that replicate via a double-

stranded DNA intermediate. The infection cycle involves the
precise integration of this intermediate into the genome of the
host cell, where it is transcribed to yield daughter genomes
that are packaged into virions.