Molecular Life Sciences

DOI 10.1007/978-1-4614-6436-5_87-1
# Springer Science+Business Media New York 2014

Bacteriophage and Viral Cloning Vectors

Douglas Julin*
Department of Chemistry and Biochemistry, University of Maryland, College Park, MD, USA

Synopsis
Viruses and bacteriophages (the viruses that infect bacteria) provided vectors for some of the first
cloning experiments, and they continue to be used in a variety of applications. The most important
bacteriophages have been the filamentous phages (especially M13), bacteriophage lambda, and
bacteriophage P1. In addition, vectors such as phagemids and cosmids have been constructed that
combine features of both plasmids and phage. A large variety of vectors can be used to generate
infectious recombinant viruses for use in eukaryotic cells. These include viruses derived from
human immunodeficiency virus type 1 (HIV-1), the causative agent of AIDS.

Introduction
Some of the earliest cloning experiments made use of bacteriophages or viruses to carry recombinant
DNA molecules, introduce them into cells, and enable the production of new phage or viral particles
containing copies of the recombinant DNA. Since then, many cloning vectors derived from natural
bacteriophages and viruses have been developed for use in a wide variety of applications.
Viruses and bacteriophage consist of, minimally, a nucleic acid genome (DNA or RNA) enclosed
within a protein capsid. Some viruses are enclosed within a lipid membrane envelope. The virus or
bacteriophage enters an appropriate host cell where the genome can be replicated and viral- or
phage-specific proteins expressed. New virus or phage particles are produced, which leave the
original cell to infect other cells. Bacteriophages, the viruses that infect bacteria, are thought to be the
most abundant “organisms” on earth (Krupovic et al. 2011).

Main Text
Cloning Vectors Derived from Bacteriophages
Cloning vectors have been developed from filamentous bacteriophages (M13 and f1), bacteriophage
lambda, and bacteriophage P1, taking advantage of the unique characteristics of each of these
phages. Bacteriophages, commonly called phages, have several advantages over plasmids as
cloning vectors. The phage infection process is an easy and highly efficient way to introduce
recombinant molecules into host cells. Infected cells can be spread on an agar plate, on which the
cells grow to produce a continuous lawn of bacteria except for the small region surrounding an
infected cell. The phage produced by the initially infected cell will infect neighboring cells, either
killing them or inhibiting their growth. This results in formation of a clear area in the bacterial lawn,
called a plaque. Formation of the plaque readily indicates the presence of the phage vector in that
region of the agar plate. High infection or transformation efficiencies of phage increase the chance of

*Email: djulin@umd.edu

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recovering a clone containing the desired recombinant DNA. Some phage vectors can hold larger
inserts than most plasmids, allowing the cloning of large eukaryotic genes and their regulatory
elements. The larger insert size also reduces the total number of clones needed for a DNA library to
contain the entire genome from a species.

Filamentous Phage (M13 and f1) and Phagemids

Joachim Messing developed a cloning system based on the M13 phage in the late 1970s (Messing
1983, 1991). These phages are useful because their genome can exist and be isolated in either
circular double-stranded (dsDNA) or single-stranded (ssDNA) forms. The circular single-stranded
recombinant DNA can be used in some DNA sequencing and site-directed mutagenesis applications
(although these approaches have been superceded by newer methods that use dsDNA). The phage
particles can also be used to make strand-specific ssDNA probes for Southern blotting. These
vectors are also important historically in the development of cloning technology (Messing 1991).
M13 and other filamentous phage particles consist of a circular single-stranded genome encased
in a capsid of about 2,700 monomers of the phage-encoded gene 8 protein (gp8) (Kornberg and
Baker 1991). The phage particle is a narrow filament that is about 895 nm long but only 6 nm in
diameter. The phage infects cells by binding to the F pilus formed by proteins encoded by the
F plasmid (fertility factor) that must be present in the host cell. Once inside the cell, the 6,407 nt
genome is converted to a circular double-stranded form called replicative form I (RFI) (Fig. 1). RFI
serves as a template for production of circular single-stranded viral DNA molecules, which are

Fig. 1 Bacteriophage M13 life cycle. Phase 1: the single-stranded circular phage DNA (+) strand enters the bacterial
cell upon infection. It is converted to a nicked double-stranded form after RNA primer synthesis by the host RNA
polymerase and DNA polymerase III. The nicked form is converted to the closed-circular supercoiled replicative form
I (RFI) by the host DNA polymerase I, ligase, and gyrase. The (
) strand in RFI is the template for phage mRNA
synthesis by the host RNA polymerase. Phase 2: the gene 2 protein encoded by the phage (gp2) nicks the RFI and
initiates synthesis of new single-stranded circular (+) strands. These new single-stranded circles are used for synthesis of
more RFI molecules early in infection. Phase 3: later in infection, the gp5 protein (red) binds the single-stranded (+)
strand products and prevents them from being replicated to RFI. Instead, gp5 is displaced by gp8 (green) to form new
phage particles that are extruded through the cell membrane. Phage proteins gp3, gp6, gp7, and gp9, that are present in
the phage particle, are not shown (After Kornberg and Baker 1991)

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themselves converted to more RFI molecules. RFI also provides the template for phage mRNA
production, allowing synthesis of phage proteins. As phage proteins accumulate in the infected cell,
the new ssDNA circles are coated by the gene 5 protein (gp5) which prevents them from being
converted to RFI and instead shunts them to packaging into phage particles. Packaging involves
displacement of gp5 by gp8, the capsid protein. Gp8 resides in the bacterial inner membrane, so
packaging is concomitant with extrusion of the gp8-DNA phage particle through the cell membrane
and to the surrounding medium. The process continues until the ssDNA is completely coated by gp8
and extruded, regardless of the size of the DNA. Thus, recombinant phage can be formed with fairly
large DNA inserts. The extrusion process does not kill the infected cell, although the infection
process slows cell growth. Thus, plaques formed by infected cells can be seen as areas of slower
bacterial growth in the surrounding bacterial lawn. Recombinant phage can be produced in large
amounts by picking a plaque from the lawn and growing the phage in liquid culture containing host
cells. The phage particles that are released into the growth medium can be isolated by precipitation
and centrifugation and the circular ssDNA isolated by phenol extraction (Messing 1983). The
double-stranded RFI can be isolated from infected cells by methods used for plasmid DNA isolation.
Messing introduced many novel features into the natural M13 phages, producing the M13mp
series of vectors, of which the most important are M13mp18 (Fig. 2) and M13mp19. These features
include a multicloning site (MCS) with several adjacent and unique restriction enzyme cleavage
sites from the plasmid pUC18, and the E. coli lac promoter and a downstream lacZa gene fragment.
Cloning into these vectors is done by isolating the double-stranded RFI form from infected cells and
ligating insert DNA into appropriate restriction sites in the MCS. The recombinant circular DNA is
transformed into host cells where the dsDNA is replicated by the normal process, to produce phage
particles containing the circular ssDNA recombinant genome. The lacZa gene allows recombinant
phage to be isolated by blue-white screening.
A novel extension of the use of filamentous phage vectors was the development of phagemid
vectors. Phagemids combine the ssDNA production of filamentous phage vectors with the simplicity
of plasmids. A phagemid contains a plasmid origin of replication and the phage sequences from M13
or f1 that are required to initiate circular ssDNA production (Mead et al. 1986). The recombinant
phagemid can be maintained and propagated in a cell as a plasmid. Phagemid ssDNA can be
produced by coinfecting cells with the phagemid and a “helper” phage or a plasmid that carries

Fig. 2 Map of M13mp18. Thick line is M13 DNA. Thin line is DNA from the E. coli lac operon. The locations of the
origin of replication region of M13, the lacZa gene (for blue-white screening), and the multicloning site (MCS) are
shown. Based on sequences in GenBank (Accession numbers M77815 and J02465)

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the genes encoding phage proteins required for ssDNA production. The phagemid is then replicated
and packaged as a phage which can be isolated and processed as above.

Bacteriophage Lambda
Peter Lobban, while a graduate student at Stanford University, proposed in 1969 to use bacterio-
phage lambda as a cloning vector by replacing a nonessential portion of the phage DNA with insert
DNA from another source (Berg and Mertz 2010). One of the first applications of this approach was
by Murray and Murray, who created a lambda phage with a portion of the nonessential region of its
genome deleted and only one EcoRI restriction site in the entire vector. Using this vector they were
able to clone a DNA fragment containing part of the E. coli trp operon genes (Murray and Murray
1974). At about the same time, Ron Davis and co-workers produced a recombinant lambda phage
carrying DNA from Drosophila melanogaster (Thomas et al. 1974).
Bacteriophage lambda has an icosahedral-shaped proteinaceous head that contains the DNA
genome and a tail that enables the phage to attach to the E. coli host cell (Kornberg and Baker
1991). The phage infects the cell by binding to the LamB protein, a maltose-binding receptor protein
in the E. coli outer membrane, and then injecting its DNA genome into the cell. For this reason, host
cells must be grown on medium containing maltose. The lambda genome is a linear double-stranded
DNA molecule of 48 kb with 12 nucleotide single-stranded overhangs on the 50 -ends. The two
overhangs, called cohesive ends, are complementary which enables the DNA to circularize by base
pairing inside the infected cell (Fig. 3). The DNA circle is sealed by the host DNA ligase and DNA
gyrase introduces negative supercoils (Fig. 4). The circularized DNA molecule serves as the
template for DNA replication and for transcription, leading to expression of phage proteins.
A lambda phage infection can take either a lysogenic or lytic course. The lysogenic phase is when
the phage DNA is inserted into the host cell genome by the action of the Int protein, a phage-encoded
protein that acts as a site-specific recombinase. The lytic phase involves production of several
hundred progeny phages which lyse and kill the host cell and are thereby released and able to infect
other cells. The choice between lysogenic and lytic growth depends on three transcription regulatory
proteins, the cI repressor (lambda repressor), cII repressor, and Cro repressor.
In lytic growth (Fig. 4), phage-encoded proteins are produced and the phage DNA is replicated.
Replication depends on host enzymes, except for the phage-encoded O and P proteins that act in
replication initiation to direct the host replication machinery to the viral genome. Replication occurs

Fig. 3 The cos site of bacteriophage lambda. The double-stranded cos sequence is in red. Cleavage of the site in linear
phage lambda DNA concatemers by the terminase enzyme produces the 12-nt cohesive ends that are found in the linear
phage genome. The host DNA ligase catalyzes joining of the cohesive ends in the monomer-length lambda genome to
produce the circular form in the infected cell (see Fig. 4)

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Fig. 4 Bacteriophage lambda lytic life cycle. The double-stranded lambda genome is injected into a host cell (top, left).
The cos cohesive ends enable the linear genome to circularize (see Fig. 3). The host DNA ligase and gyrase produce
a supercoiled genome. The lambda O protein binds to the replication origin and the P protein recruits host replication
enzymes, to initiate replication via a theta structure intermediate. This process produces more supercoiled genomes.
Later in infection, the replication switches to a rolling circle mechanism that produces linear concatemers of the phage
genome. The linear DNA can enter phage proheads as they assemble in the cell. When the head is completely filled with
one genome length of DNA, terminase cleaves two cos sites, to release a monomer-length genome in the phage head.
Phage particles are completed by addition of the tail and released to the surroundings after cell lysis. The thick red line
represents double-stranded DNA, in which individual strands are not shown (After Kornberg and Baker 1991)

initially by a theta-type mechanism, producing closed-circular product DNA. About 15 min after
initiation, the replication switches to a rolling circle mechanism that produces linear double-stranded
DNA concatemers in which monomer-length genomes are joined in a head-to-tail fashion. The
concatemeric genome is necessary for packaging of monomer-length genomes into the phage
proheads that assemble in the infected cell. Packaging requires cleavage of monomer genomes
from the concatemer by the phage-encoded terminase endonuclease. Terminase cleaves the DNA at
the cos site to regenerate the 12-nt cohesive ends of the original phage DNA.
Lytic infection requires genes on both ends of the lambda phage’s linear genome. However, the
DNA between these two “arms,” about one-third of the genome, is not essential for phage
propagation and can be replaced with insert DNA (Fig. 5). Cloning involves replacing the central
nonessential “stuffer fragment” by ligating insert DNA between the two arms, packaging the
recombinant DNA into phage particles in vitro and infecting E. coli cells with the recombinant
phage particles. Phage replication in the host cell produces dsDNA molecules consisting of repeated
recombinant phage DNA joined at cos sites. Terminase processing cleaves the DNA at the cos sites,
allowing packaging of the recombinant DNA into phage particles that eventually lyse the cell,

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Fig. 5 Bacteriophage lambda genome map. The locations of the genes mentioned in the text are indicated, including the
genes that encode the small and large subunits of terminase (Nu1 and A gene), the O and P genes encoding phage-
specific DNA replication proteins, and the cI, cII, and cro genes involved in the choice of lysogenic or lytic growth. The
DNA between the J and N genes is dispensable and can be replaced by a DNA insert when the phage is used as a cloning
vector. The replaceable region includes genes encoding enzymes for integration and excision of the prophage (int and
xis) and for homologous recombination (exo, bet, and gam), among others. See (Sambrook et al. 1989) for a more
complete map. Map coordinates are from the sequence in GenBank, accession # NC_001416

releasing the phage. The amount of DNA that enters the prohead must be enough to fill the limited
volume of the head and thus must be between 75 % and 105 % of the normal length of the phage
genome (Kornberg and Baker 1991; Chauthaiwale et al. 1992; Weigel and Seitz 2006). This limits
both the minimum and maximum sizes of the DNA insert. Lambda insertion vectors allow cloning of
small inserts into the nonessential region, while lambda replacement vectors remove the entire
nonessential region and allow inserts to around 23 kb in length (Chauthaiwale et al. 1992). Lambda
phage vectors can be purchased as individual arms produced by restriction endonuclease cleavage,
ready to be ligated to insert DNA that was cut with the same enzyme. E. coli cell extracts containing
the enzymes required for packaging are also available, so that the recombinant phage DNA can be
packaged in vitro into viral particles.
A number of improved lambda phage vectors have been developed (Chauthaiwale et al. 1992), by
adding multicloning sites for cloning insert DNA, promoters for gene expression, and the lacZa gene
fragment for blue-white selection capability. For example, the lgt11 vector (Young and Davis 1983)
contains the lac promoter and the lacZ gene. DNA up to 7.2 kb in size can be inserted into a unique
EcoRI site within the lacZ coding sequence. Recombinant plaques can be screened for production of
the desired protein as a LacZ fusion protein by using an antibody directed against the target protein.

Vectors Based on Bacteriophage Lambda: Cosmids and Lambda ZAP

Cosmid vectors were developed in the late 1970s (Collins and Hohn 1978). These vectors are small
plasmids (~5 kb) that contain (minimally) a plasmid origin of replication and antibiotic resistance
gene and the cos site from bacteriophage lambda. The cos site allows the recombinant plasmid DNA
to be cleaved by lambda terminase and packaged into phage particles which can be introduced very
efficiently into host cells by the phage infection process (Collins and Hohn 1978). Cosmids can
accommodate DNA inserts of 35–45 kb, larger than are easily handled using ordinary plasmid
vectors. Cosmids have been particularly useful for generating DNA libraries.
DNA can be inserted into a cosmid vector by cleavage with an appropriate restriction enzyme,
followed by ligation under conditions where the ligation products are linear concatemers rather than
circles. The concatemers contain multiple copies of the linear cosmid vector ligated to and separat-
ing random insert DNA molecules. The cos sequences in these concatemers enable the recombinant
molecules to be packaged into phage particles. The ligation mixture is mixed with a cell extract that
contains phage proheads, tails, and packaging enzymes, including the terminase enzyme. Terminase
cleaves the linear DNA concatemers at the cos sites and the resulting linear products are packaged as

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phage. The phages are then used to infect E. coli cells. The cohesive ends from the cleaved cos sites
in the recombinant linear DNA enable the DNA to be circularized and converted to a plasmid in the
cell (Fig. 3), where it is subsequently replicated using the plasmid replicon. Phages are not produced
in these cells because the cosmid lacks all lambda genes that are needed for producing phage
particles and for cell lysis. Vectors with small or no DNA insert are too small to be packaged into
phage heads, and so these molecules are selected against.
The lambda ZAP vector combines features of phage lambda, filamentous phage f1, and the
Bluescript plasmid, giving a vector that provides high efficiency of transformation, simplicity of
storage, ease of screening a lambda phage library, and easy excision of the insert DNA into the
pBluescript plasmid vector for further analysis (Short et al. 1988). The latter feature greatly
facilitates subcloning of DNA from the recombinant phage to the plasmid, where the DNA is in
many respects easier to manipulate than in the phage vector.
The lambda ZAP vector consists of the arms from phage lambda, between which the Bluescript
phagemid has been inserted. Bluescript has both a plasmid (pUC) replicon and that from the f1
filamentous phage. Foreign DNA is ligated into the Bluescript portion of the lambda ZAP vector,
and the resulting recombinant molecules are packaged into lambda phage and used to infect an
E. coli host. The Bluescript plasmid and cloned DNA insert can be excised in circular double-
stranded form by introducing the recombinant lambda ZAP phage into cells that express f1 phage
proteins required for replication of the f1 replicon contained within pBluescript. The recombinant
DNA is maintained thereafter as a circular plasmid. The f1 replicon can also be used to generate the
circular single-stranded form of the recombinant Bluescript plasmid. The pBluescript insert in
lambda ZAP has a multicloning site for convenient insertion of foreign DNA, and the lac promoter
and lacZa, allowing for blue-white selection using X-gal.

Bacteriophage P1
Vectors based on bacteriophage P1 were developed in the early 1990s (Sternberg 1990). These
vectors can accommodate very large DNA inserts (ca. 100 kb) without the instability problems that
were encountered with yeast artificial chromosome (YAC) vectors. Recombinant DNA in P1 vectors
can be introduced into cells with high transformation efficiency by the phage infection process.
The P1 bacteriophage particle has a linear 94.8 kb double-stranded DNA genome (Lobocka et al.
2004). The phage can pursue both lytic and lysogenic phases in an infected cell. The phage exists as
a circular plasmid when in the lysogenic phase. The plasmid form is replicated as the cell undergoes
replication and division, and it is maintained at one to two copies per cell by a phage-encoded
partition system. Replication of the circular prophage requires an origin of replication (oriR) and the
phage-encoded repA origin-binding protein, along with several host enzymes (Kornberg and Baker
1991).
Circularization of the linear phage genome after entry into the host cell depends on the Cre-lox
site-specific recombination system. The phage-encoded Cre protein can bind, cleave, and rejoin two
loxP sequences in double-stranded DNA. The linear phage genome has a terminal redundancy in
which about 9–12 % of the genome sequence is repeated at the ends of the genome (Kornberg and
Baker 1991). If the repeated region happens to include the loxP site, then cleavage and joining of the
two sites by Cre produces the circular plasmid form of the phage.
P1 cloning vectors (Sternberg 1990) contain the pBR322 replicon that allows them to be
maintained as multi-copy plasmids prior to insertion of foreign DNA. The vectors also contain the
P1 packaging site (pac) required for packaging of recombinant DNA into the P1 phage head in vitro.
Packaging of DNA continues until the head is filled, at which point the DNA is cleaved by the
phage-encoded pacase enzyme present in the in vitro packaging extract. The amount of DNA that is

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packaged (110–115 kb) is determined by the amount of the DNA required to fill the icosahedral
phage head. The recombinant P1 phages are then used to infect an E. coli cell that expresses the Cre
recombinase. The recombinant DNA is circularized and maintained in the cell as a single copy by the
P1 plasmid replicon in the vector, since the pBR322 replicon is removed during circularization. The
P1 lytic replicon in the recombinant molecule is under transcriptional control of the lac operon.
Expression of the lytic genes, which increases the copy number of the plasmid 25-fold, can be
induced by adding IPTG to relieve repression by the Lac repressor. The increase of the plasmid copy
number aids isolation of the recombinant DNA.

Vectors Derived from Eukaryotic Viruses

Many vectors have been developed by modifying natural viruses for convenient use in mammalian
cells, much as was done with bacteriophage vectors described above. Vectors have been based on
retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, and others (Blesch 2004;
Chailertvanitkul and Pouton 1992; Deyle and Russell 2009; McConnell and Imperiale 2004;
Quinonez and Sutton 2002). A useful vector includes viral sequences that are essential for stable
replication and selection in a mammalian host cell and for packaging into a virion particle. The
vector also has sequences that allow it to be maintained as a circular plasmid in a bacterial host. DNA
is cloned into the vector by standard cloning techniques using E. coli as the host. The recombinant
vector is then introduced into an appropriate eukaryotic host cell that has been engineered to produce
the viral proteins required for packaging the viral DNA (or RNA) genome into virion particles.
Finally, the virus particles that now contain the recombinant vector with DNA insert are introduced
into the host cell of interest, where the inserted DNA can be expressed.
These vectors can be used in several applications, including to introduce genes into mammalian
cells for study of the biological effect of expression of that gene or for gene therapy. Induced
pluripotent stem cells have been generated from somatic cells by introducing genes encoding key
regulatory proteins carried on virus vectors (Patel and Yang 2010). Baculovirus-based vectors are
used to achieve high-level expression of active proteins from eukaryotic sources, in eukaryotic
(insect) host cells. Plasmid (episome) vectors capable of stable replication in mammalian cells have
been developed from natural viruses (see the Short Essay “▶ Plasmid Cloning Vectors”).

Vectors Based on Retroviruses and Lentiviruses

Retroviruses and lentiviruses have single-stranded RNA genomes. The RNA serves as the mRNA
for viral protein synthesis, and it is converted to dsDNA by reverse transcription in the infected cell.
The dsDNA can then be integrated into the host cell chromosome, giving a cell line that is stably
transfected by the virus. The Moloney murine leukemia virus was one of the first important retroviral
vectors (Quinonez and Sutton 2002). A number of lentiviruses have been used more recently,
including human immunodeficiency virus (HIV), the causative agent of AIDS. Lentivirus-based
vectors are advantageous because the virus can infect both dividing and nondividing cells (Lois et al.
2002).
Retroviral and lentiviral vectors contain sequences required for maintenance as a dsDNA plasmid
in E. coli and (minimally) the viral 50 - and 30 -long terminal repeat sequences (LTRs) that are required
for viral RNA reverse transcription in a human host cell and the viral Psi (C) packaging signal
sequence (Fig. 6). Insert DNA is ligated into restriction sites placed between the LTRs, under the
control of a promoter such as that from cytomegalovirus (CMV). The recombinant plasmid version
of the vector is transfected into a packaging cell line. These cells contain plasmids with the genes
encoding proteins necessary for viral RNA production and packaging into viral particles with
a protein capsid and membrane envelope. The virion particles contain the viral RNA and the reverse

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Fig. 6 Examples of vectors derived from a lentivirus (pFUGW-H1) and retrovirus (pCX4hyg). The pFUGW-H1 vector
contains the following elements from HIV-1: 50 - and 30 -long terminal repeats (LTR) (the 30 LTR has a deletion (DU3) that
inactivates transcription of the integrated provirus from the 50 LTR), Psi (C) region for packaging into virion particles,
and the Rev-response element (RRE) for efficient pre-mRNA splicing. Other features are the cytomegalovirus (CMV)
promoter to increase expression of the viral RNA genome during transient transfection, the enhanced green fluorescent
protein (EGFP) transgene controlled by the human ubiquitin-C (hUbC) promoter, and the woodchuck hepatitis virus
posttranscriptional response element (WPRE) for transgene mRNA stabilization. The vector also has the replication
origin and ampicillin resistance gene (Ampr) from pBR322, enabling the plasmid to be maintained in E. coli (From Lois
et al. 2002; Fasano et al. 2007) and http://www.addgene.org/25870/). The pCX4hyg vector contains the 50 and 30 LTRs,
Psi packaging signal region, and splice acceptor site elements from the Moloney murine leukemia virus (MLV). It also
contains an internal ribosome entry site (IRES) from encephalomyocarditis virus (ECMV), a gene encoding resistance to
hygromycin (Hyg), the CMV immediate early enhancer, and the ampicillin resistance gene and origin of replication from
pBR322 (From GenBank (accession # AB086387) and Akagi et al. 2003)

transcriptase and integrase enzymes that act when the recombinant viruses infect the final target host
cell. However, the recombinant viruses lack the genes encoding these proteins, as well as all other
genes necessary for producing new infectious virus in the target cells. The HIV Env protein in the
membrane envelope of the virus normally binds to the CD4 protein on the surface of T cells
(Quinonez and Sutton 2002). However, packaging cell lines are available that introduce envelope
proteins from other viruses that enable the recombinant virus to target a wide variety of cell types.
Once introduced into the target cell, the viral RNA is reverse transcribed and transported into the
nucleus. There, the viral integrase enzyme catalyzes irreversible integration of the dsDNA genome
into the host chromosome. The insert gene is then expressed from the promoter carried in the vector.

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HIV-based viruses can be used for introducing genes for functional studies and in gene therapy
applications. A potential drawback with these integrating viruses is that they might disrupt an
important gene in the target host cell, with potential adverse consequences for the cell.

Adenovirus and Adeno-associated Viruses

Adenoviruses have linear dsDNA genomes of about 36 kb. They are taken up by endothelial cells via
clathrin-mediated endocytosis and transported to the cell nucleus where viral DNA replication is
catalyzed by a viral-encoded DNA polymerase. Adenoviruses have been used for gene therapy,
although they suffer from a number of drawbacks including an intense inflammatory response and
being immunogenic (Chailertvanitkul and Pouton 1992). Thus, vectors derived from adenovirus
have most viral genes deleted, in order to minimize the immune response in a human host
(McConnell and Imperiale 2004). This allows the vector to hold a relatively large DNA insert. As
described above, the vector is first used to infect cells that produce proteins necessary for viral
packaging. The resulting virus particles are then used to infect a target cell or individual.
Adeno-associated virus (AAV) has a small (~4.7 kb) linear ssDNA genome (Deyle and Russell
2009). AAV is naturally dependent on other viruses for replication and packaging proteins. AAV
vectors typically have only the inverted terminal repeat sequences, between which the foreign DNA
is inserted. Recombinant viruses are produced by infecting helper cells and then used to infect
a target host cell. The viral DNA enters the nucleus of the infected cell, where it can exist as a circular
episome or integrate into the cell chromosome. Integration is rare, occurring with only about 0.1 %
of the viruses that enter cells.

Baculovirus Vectors
Baculovirus vectors that allow for high-level expression of cloned genes in eukaryotic cells (insect
cells) were developed in the early 1980s. They are useful for the expression of proteins from
mammalian and other eukaryotic sources, since the proteins undergo correct posttranslational
modification, such as glycosylation, in the insect cell host (Miller 1989; Smith et al. 1983). These
modifications do not occur when the protein is expressed in bacterial cells.
Baculoviruses have large (80–180 kb) dsDNA genomes (Miller 1989). They infect lepidopteran
insects, such as Autographa californica (alfalfa looper), Bombyx mori (silkworm), and Spodoptera
frugiperda (fall armyworm). The viral life cycle involves two stages. New viruses are formed and
bud from the membrane of an infected cell, ca. 10–24 h’ postinfection. These viruses travel to and
infect new cells in the infected organism. Later (ca. 18–70 h’ postinfection), membrane-enveloped
virus particles accumulate in the nucleus of the cell in the form of viral occlusions. The occlusions
include large amounts of a highly expressed viral protein called polyhedrin. Polyhedrin and a second
viral protein called P10 are expressed at very high levels late in infection by the baculovirus.
Neither the polyhedrin nor the P10 protein is required neither for viral infection of a host cell nor
for production of infectious progeny viruses. Therefore, the DNA encoding either of these proteins
can be replaced by foreign insert DNA. The insert DNA is then downstream of the strong polh or p10
promoter, and the foreign gene is expressed at high levels. This is the basis of using baculoviruses for
expressing foreign proteins. Baculoviruses are rod shaped and normally contain 80–200 kb of
dsDNA. The viral rod can expand to accommodate recombinant viruses containing large inserts,
similar to filamentous bacteriophages.
The size of the viral genome makes it impractical to insert DNA by simple ligation of DNA
fragments. Instead, the insert DNA is first cloned into a transfer plasmid that is propagated in and
isolated from E. coli cells. The transfer plasmid contains the promoter from either the polyhedrin
gene (polh) or the P10 gene (p10). The promoter is flanked on both sides by viral DNA. The foreign

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Fig. 7 Construction of a recombinant baculovirus by homologous recombination. The gene to be expressed (yellow) is
first cloned into a transfer plasmid by standard techniques in bacterial cells. The gene is downstream of the strong p10
promoter from baculovirus and flanked by the lef2 and orf1629 genes from the virus. The transfer plasmid is
co-transfected into insect cells along with a linearized baculovirus genome with a truncated form of orf1629. Recom-
bination between the homologous viral sequences in the two DNA molecules generates a complete recombinant
baculovirus, with the DNA insert and p10 promoter. The recombinant baculovirus also gains a complete copy of
orf1629, an essential viral gene, which facilitates selection of cells containing recombinant virus. Other variations are
available (see van Oers 2011). Figure is based on van Oers (see van Oers 2011). Transfer plasmid and baculovirus are not
drawn to the same scale

DNA is inserted downstream of the promoter and within the flanking DNA by standard ligation
methods. The recombinant transfer plasmid and baculovirus DNA are co-transfected into insect cells
(Sf9 or Sf21 cell). Homologous recombination between the plasmid and the virus is catalyzed by the
cellular recombination machinery (Fig. 7). The flanking DNA in the targeting plasmid replaces the
corresponding viral DNA after crossovers on both sides of the DNA insert.
The first application of baculovirus was for the expression of human beta interferon (Smith et al.
1983). The beta interferon gene replaced the polyhedrin gene in the recombinant virus. The
interferon protein was produced in large amount, secreted from the cells as expected, and
glycosylated.
Several modifications to the baculovirus system have made the cloning and screening procedures
more convenient. The bacmid system was developed to simplify and streamline the process of
obtaining recombinant virus containing the desired DNA insert (Luckow et al. 1993). The DNA
insert is first ligated into a transfer plasmid, as described above. The transfer plasmid contains the
polyhedrin gene promoter flanked by the left and right ends of the Tn7 transposon rather than by
baculovirus DNA. The plasmid vector also contains the lacZa gene downstream of the polyhedrin
promoter and a gentamicin resistance gene. The baculovirus is in the form of a shuttle vector, called
a bacmid, that contains a mini-F replicon that can be replicated in E. coli and viral sequences
required for virus production in insect cells. The bacmid also contains the attachment site for the Tn7
transposon (attTn7) and a kanamycin resistance gene. The transfer plasmid and bacmid are
introduced into E. coli cells, along with a helper plasmid that encodes the Tn7 transposase
(TnsABCD genes). The transposase catalyzes transposition of the mini-Tn7 (containing the insert
DNA downstream of the polyhedrin promoter) into the bacmid. The recombinant bacmid can then
be introduced into insect cells, where it replicates and expresses protein encoded by the inserted
gene. The LacZa gene allows for blue-white screening for recombinant bacmids.
The efficiency of recombinant virus isolation was improved by adding the sacB gene from
Bacillus subtilis to the transfer plasmid used in the bacmid system, to make the pBVboost vector
(Airenne et al. 2003). SacB encodes the enzyme levansucrase, which produces a toxic product in
E. coli when the cells are grown in the presence of sucrose. E. coli cells that still contain intact
transfer plasmid after the Tn7-mediated transposition step, presumably because the transposition

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DOI 10.1007/978-1-4614-6436-5_87-1
# Springer Science+Business Media New York 2014

failed in that cell, are killed by growth on sucrose. This negative selection step results in greater ease
of isolating desired recombinant baculoviruses (bacmids).

Cross-References
▶ Artificial Chromosomes
▶ Blue/White Selection
▶ Plasmid Cloning Vectors
▶ Recombineering

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