Vectors bacteriophages

1. Amjad Khan Afridi

2. PHAGEPHAGE
VECTORSVECTORS
.

3. PHAGESPHAGES
• Derivatives of phage have been developed as cloning vectors since the earlyDerivatives of phage have been developed as cloning vectors since the early
days of gene technology.days of gene technology.
• The phage derivatives are considered to be the most suitable cloning vehiclesThe phage derivatives are considered to be the most suitable cloning vehicles
for cloning genomic eukaryotic DNA because of the following advantages overfor cloning genomic eukaryotic DNA because of the following advantages over
the plasmids.the plasmids.
 Thousands of phage plaques can be obtained in a single petri dish.Thousands of phage plaques can be obtained in a single petri dish.
 Selection bySelection by DNA-DNA hybridizationDNA-DNA hybridization is possible.is possible.
 In vitro packagingIn vitro packaging into empty phage head is possible thus increasing phageinto empty phage head is possible thus increasing phage
infectivityinfectivity
Size selection of the packaged DNA is possible.Size selection of the packaged DNA is possible.
 Millions of independently cloned virus particle can be constituted to form aMillions of independently cloned virus particle can be constituted to form a
gene library.gene library.
.

4. BACTERIOPHAGESBACTERIOPHAGES • Bacteriophage is aBacteriophage is a genetically complexgenetically complex butbut
very extensively studied virus ofvery extensively studied virus of E.coliE.coli..
• The DNA of phage, is aThe DNA of phage, is a linear duplex moleculelinear duplex molecule
of 48502 bpof 48502 bp (~49kb) in length.(~49kb) in length.
• The DNA isolated from virus particles is aThe DNA isolated from virus particles is a
double stranded linear molecule with shortdouble stranded linear molecule with short
complementary single stranded projections ofcomplementary single stranded projections of
12 nucleotides at its 5’ ends.12 nucleotides at its 5’ ends.
• These cohesive termini, also referred to asThese cohesive termini, also referred to as coscos
sites,sites, allow the DNA to be circularizedallow the DNA to be circularized afterafter
infection of the host cell and also packaging ofinfection of the host cell and also packaging of
the DNA.the DNA.
.

5. GENERAL STRUCTURE OF BACTERIOPHAGE DNAGENERAL STRUCTURE OF BACTERIOPHAGE DNA
• The genetic map of phage comprises approximately 40 genes which areThe genetic map of phage comprises approximately 40 genes which are
organized in functional clusters.organized in functional clusters.
• Genes coding for head and tail are proteins (genesGenes coding for head and tail are proteins (genes A-JA-J) are on the left of the) are on the left of the
linear map.linear map.
• The central region contains genes, such asThe central region contains genes, such as int, xis, exoint, xis, exo etc., which areetc., which are
responsible forresponsible for lysogenisationlysogenisation i.ei.e the process leading to the integration of viralthe process leading to the integration of viral
DNA and other recombination events.DNA and other recombination events.
• Much of this central region is not essential for lytic growth.Much of this central region is not essential for lytic growth.
• Genes to the right of the central region compriseGenes to the right of the central region comprise six regulatory genessix regulatory genes, two, two
genes (genes (OO andand PP) which are essential) which are essential for DNA replicationfor DNA replication during lytic growth andduring lytic growth and
two more genes (two more genes (SS andand RR) which are required) which are required for the lysis of the cellularfor the lysis of the cellular
membranes.membranes.
.

6. • In the phage DNA, larger central region is not essential for phage growth andIn the phage DNA, larger central region is not essential for phage growth and
replication.replication.
• This region of phage can be deleted or replaced without seriously impairingThis region of phage can be deleted or replaced without seriously impairing
the phage growth cycle.the phage growth cycle.
• Using this non-essential region of phage, several phage vector derivatives haveUsing this non-essential region of phage, several phage vector derivatives have
been constructed for efficient gene cloning.been constructed for efficient gene cloning.
.

7. TYPES OF PHAGE VECTORSTYPES OF PHAGE VECTORS
• Wild type phage DNA itself cannot be used as a vector since it contains tooWild type phage DNA itself cannot be used as a vector since it contains too
many restriction sites.many restriction sites.
• Further, these sites are often located within the essential regions for phage'sFurther, these sites are often located within the essential regions for phage's
growth and development.growth and development.
• From these wild phages, derivatives with single target sites and two targetFrom these wild phages, derivatives with single target sites and two target
sites have been synthesized.sites have been synthesized.
• Phage vectors whichPhage vectors which contain single sitecontain single site for the insertion of foreign DNA havefor the insertion of foreign DNA have
been designated asbeen designated as Insertional vectorsInsertional vectors;;
• vectors withvectors with two cleavage sitestwo cleavage sites, which allow foreign DNA to be substituted for, which allow foreign DNA to be substituted for
the DNA sequences between those sites, are known asthe DNA sequences between those sites, are known as replacement vectorsreplacement vectors..
.

8. INSERTIONAL VECTORSINSERTIONAL VECTORS
• A large segment of the non-essential region hasA large segment of the non-essential region has
been deleted, and the two arms ligated together.been deleted, and the two arms ligated together.
• An insertion vector possesses at least one uniqueAn insertion vector possesses at least one unique
restriction site into which new DNA can be inserted.restriction site into which new DNA can be inserted.
• Two popular insertion vectors are:Two popular insertion vectors are:
• Egt10 :Egt10 : which can carrywhich can carry up to 8 kbup to 8 kb of new DNA,of new DNA,
inserted into ainserted into a uniqueunique EcoEcoRIRI site located in thesite located in the ccII
gene.gene.
• Insertional inactivation of this gene means thatInsertional inactivation of this gene means that
recombinants are distinguished as clear rather thanrecombinants are distinguished as clear rather than
turbid plaques.turbid plaques.
• EZAPIIEZAPII : insertion of up to 10 kb DNA into any of 6: insertion of up to 10 kb DNA into any of 6
restriction sites withinrestriction sites within a polylinkera polylinker inactivates theinactivates the
lacZlacZ gene carried by the vector.′ gene carried by the vector.′
• Recombinants give clear rather than blue plaquesRecombinants give clear rather than blue plaques
on X-gal agar.on X-gal agar.
.

9. E.COLI/E.COLI/λλ REPLACEMENT VECTORSREPLACEMENT VECTORS
ExamplesExamples:: EMBL3 andEMBL3 and λλ DASH.DASH.
A representative scheme for cloning:A representative scheme for cloning:
1. The1. The λλ vector DNAvector DNA is cleaved withis cleaved with
BamH1BamH1 and the long (19 kb) andand the long (19 kb) and
short (9 kb) arms are purified;short (9 kb) arms are purified;
2. The2. The targettarget fragments are preparedfragments are prepared
by digestion, also withby digestion, also with BamBamH1 or aH1 or a
compatible enzyme (compatible enzyme (SauSau3A);3A);
3. The target fragments are treated3. The target fragments are treated
withwith alkaline phosphatasealkaline phosphatase to preventto prevent
them ligating to each other;them ligating to each other;
4. The4. The λλ armsarms and theand the targettarget
fragmentsfragments are ligated together atare ligated together at
relatively high concentration to formrelatively high concentration to form
long linear products.long linear products.
B B
∼ 20kb
B
Can not
Parking
infect
E.coli
Long armLong arm
ShortShort
armarmReplace.
48.5 kb
.

10. PACKAGING AND INFECTIONPACKAGING AND INFECTION
TheThe RecombinantsRecombinants that canthat can notnot be packaged:be packaged:
1. Ligated1. Ligated λλ ends which do not contain an insert;ends which do not contain an insert;
2. The insert is much smaller or larger than the 20 kb;2. The insert is much smaller or larger than the 20 kb;
3. The recombinants with two left or right arms.3. The recombinants with two left or right arms.
in vivo
B
Replication→ concata-mers
cleave individual λ genomes
in vitro
A mixture of phage coat proteins and
the phage DNA-processing enzymes
Packaging:
Packaging
phage
particles
Infection of E. coli
109
recombinants per mg of vector DNA..

11. FORMATION OF PLAQUESFORMATION OF PLAQUES
Plaques are the analogs of single bacterial colonies.Plaques are the analogs of single bacterial colonies.
FormationFormation::
The infectedThe infected E.coliE.coli cells from a packaging reaction are spread oncells from a packaging reaction are spread on
an agar plate,an agar plate,
The plate has been pre-spread with uninfected cells, which willThe plate has been pre-spread with uninfected cells, which will
grow to form a continuous lawn.grow to form a continuous lawn.
After incubation, phage-infected cells result in clear areas, thatAfter incubation, phage-infected cells result in clear areas, that
are plaques, where cycles of lysis and re-infection haveare plaques, where cycles of lysis and re-infection have
prevented the cells from growing.prevented the cells from growing.
Recombinant λ DNA may be purified:
• from phage particles isolated from plaques or
• from the supernatant of a culture infected
with a specific recombinant plaque.
E.coli lawn
Plaques
.

12. RF
BACTERIOPHAGE M13BACTERIOPHAGE M13
Genome features:Genome features: Size is small (6.7 kb); Single-stranded;Size is small (6.7 kb); Single-stranded;
Circular genome; DNA; Positive-sense.Circular genome; DNA; Positive-sense.
g3pg6p
g7p
g8p
g9p
Host
enzymes
end
ini
Infection: M13 particles attach specifically to E.coli sex pili (encoded by a
plasmid called F factor), through a minor coat protein (g3p). Binding of g3p
induces a structural change in the major capsid protein. This causes the whole
particle to shorten, injecting the viral DNA into the host cell.
.

13. E.COLIE.COLI/M13 PHAGE/M13 PHAGE
VECTORSVECTORS
StructureStructure:: ♣♣ TheThe phage particlesphage particles
containcontain a 6.7 kb circulara 6.7 kb circular ssDNAssDNA.. ♣♣
After infection of a sensitiveAfter infection of a sensitive E. coliE. coli
host, the complementary strand ishost, the complementary strand is
synthesized, like asynthesized, like a plasmidplasmid, and the, and the
DNA replicated as aDNA replicated as a dsDNAdsDNA, the, the
replicative form (RF).replicative form (RF).
FeaturesFeatures:: ♣♣ The host cells can continueThe host cells can continue
to grow slowly.to grow slowly.
• ssDNAssDNA: The single-stranded forms are: The single-stranded forms are
continuously packaged and releasedcontinuously packaged and released
from the cells as new phage particles.from the cells as new phage particles.
ssDNA has a number of applications,ssDNA has a number of applications,
includingincluding ♣♣ DNA sequencing andDNA sequencing and ♣♣
site-directed mutagenesis.site-directed mutagenesis.
• dsDNAdsDNA: The RF (dsDNA) can be: The RF (dsDNA) can be
purifiedpurified in vitroin vitro and manipulatedand manipulated
exactly like a plasmid.exactly like a plasmid..

14. CLONING IN M13CLONING IN M13
PurposePurpose: When the: When the single-stranded DNAsingle-stranded DNA of a fragment is required, a M 13of a fragment is required, a M 13
vector can be used as a common cloning tool.vector can be used as a common cloning tool.
PreparationPreparation of ssDNA:of ssDNA:
1.1. CloningCloning: standard plasmid cloning method can be used to incorporate: standard plasmid cloning method can be used to incorporate
recombinant DNA into M13 vectors;recombinant DNA into M13 vectors;
2.2. TransformationTransformation: the M13 then infects sensitive: the M13 then infects sensitive E. coliE. coli cells;cells;
3.3. PlatingPlating: the host cells grow to form the plaques;: the host cells grow to form the plaques;
4.4. IsolationIsolation: the ssDNA may then be isolated from phage particles in the: the ssDNA may then be isolated from phage particles in the
growth medium of the plate.growth medium of the plate.
ScreeningScreening: Blue-white screening using MCSs and lacZ' has been engineered: Blue-white screening using MCSs and lacZ' has been engineered
into M13 vectors.into M13 vectors.
ExamplesExamples: The: The M13mpl8M13mpl8 andand M13mp19M13mp19, which are a pair of vectors in which, which are a pair of vectors in which
the MCS are in opposite orientations relative to the M13 origin ofthe MCS are in opposite orientations relative to the M13 origin of
replication.replication.
.

15. HYBRID PLASMID-M13 VECTORSHYBRID PLASMID-M13 VECTORS
DefinitionDefinition: A number of small plasmid vectors, for example pBlue-script, have: A number of small plasmid vectors, for example pBlue-script, have
been developed to incorporate M13 functionality.been developed to incorporate M13 functionality.
StructureStructure: They contain both plasmid and M13 origins of replication, but do not: They contain both plasmid and M13 origins of replication, but do not
possess the genes required for the full phage life cycle.possess the genes required for the full phage life cycle.
Working waysWorking ways::
1.1. Plasmid wayPlasmid way: they normally propagate as true plasmids, and have the: they normally propagate as true plasmids, and have the
advantages of rapid growth and easy manipulation of plasmid vectors;advantages of rapid growth and easy manipulation of plasmid vectors;
2.2. Phage wayPhage way: they can be induced to produce single-stranded phage particles: they can be induced to produce single-stranded phage particles
by co-infection with a fully functionalby co-infection with a fully functional helper phagehelper phage, which provides the gene, which provides the gene
products required for single-strand production and packaging.products required for single-strand production and packaging.
.

16. COSMIDSCOSMIDS
• Cosmids areCosmids are hybrids betweenhybrids between aa phagephage DNA molecule and aDNA molecule and a
bacterial plasmidbacterial plasmid, and their design centers on the fact that, and their design centers on the fact that
the enzymes that package thethe enzymes that package the λλ DNA molecule into theDNA molecule into the
phage protein coat need only the cos sites in order tophage protein coat need only the cos sites in order to
function.function.
• The in vitro packaging reaction works not only withThe in vitro packaging reaction works not only with λλ
genomes, but also with any molecule thatgenomes, but also with any molecule that carriescarries coscos sitessites
separated by 37–52 kb of DNA.separated by 37–52 kb of DNA.
• A cosmid is basically a plasmid that carries aA cosmid is basically a plasmid that carries a coscos site .site .
• It also needs aIt also needs a selectable markerselectable marker, such as the, such as the ampicillinampicillin
resistance gene, and a plasmidresistance gene, and a plasmid origin of replicationorigin of replication, as, as
cosmids lack all thecosmids lack all the λλ genes and so do not produce plaques.genes and so do not produce plaques.
• Instead colonies are formed on selective media, just asInstead colonies are formed on selective media, just as
with a plasmid vector.with a plasmid vector.
.

17. • The following table provides a list cosmid vectorsThe following table provides a list cosmid vectors
and their structural features.and their structural features.
• Cosmid Size(kb)Cosmid Size(kb) Cleavage sites Size ofCleavage sites Size of
insertion (kb)insertion (kb)
• MUA3MUA3 4.764.76 EcoRI/PstI/PvuII/PvuIEcoRI/PstI/PvuII/PvuI 40 – 4840 – 48
• pJB8pJB8 5.405.40 BamHIBamHI 32 – 4532 – 45
• Homer I 5.40Homer I 5.40 EcoRI/ClaIEcoRI/ClaI 30 – 4730 – 47
• Homer II 6.38Homer II 6.38 SstISstI 32 – 4432 – 44
• pJC79pJC79 6.406.40 EcoRI/ClaI/BamH IEcoRI/ClaI/BamH I 32 – 4432 – 44
.

18. BACTERIAL ARTIFICIALBACTERIAL ARTIFICIAL
CHROMOSOMES (BAC)CHROMOSOMES (BAC)
BACs are based on bacterial mini-FBACs are based on bacterial mini-F
plasmids, which are small pieces ofplasmids, which are small pieces of
episomal bacterial DNA that give theepisomal bacterial DNA that give the
bacteria the ability to initiate conjugationbacteria the ability to initiate conjugation
with adjacent bacteria. They have awith adjacent bacteria. They have a
cloning limit of 75-300 kb.cloning limit of 75-300 kb.
.

19. YEAST ARTIFICIAL CHROMOSOMES (YAC)YEAST ARTIFICIAL CHROMOSOMES (YAC)
 YACs are artificial chromosomes that replicate in yeast cells. They consist ofYACs are artificial chromosomes that replicate in yeast cells. They consist of
TelomeresTelomeres, which are ends of chromosomes involved in the replication and stability, which are ends of chromosomes involved in the replication and stability
of linear DNA.of linear DNA.
 Origin of replicationOrigin of replication sequences necessary for the replication in yeast cells.sequences necessary for the replication in yeast cells.
 AA yeast centromereyeast centromere, which is a specialized chromosomal region where spindle, which is a specialized chromosomal region where spindle
fibers attach during mitosis.fibers attach during mitosis.
 A selectable marker for identification in yeast cells.A selectable marker for identification in yeast cells.
 Ampicillin resistanceAmpicillin resistance gene for selective amplification.gene for selective amplification.
 Recognition sites for restriction enzymes.Recognition sites for restriction enzymes.
.

20. THE PROCEDURE FORTHE PROCEDURE FOR
MAKING YAC VECTORS IS ASMAKING YAC VECTORS IS AS
FOLLOWSFOLLOWS
1. The target DNA is partially digested by a1. The target DNA is partially digested by a
restriction endonuclease, and the YAC vector isrestriction endonuclease, and the YAC vector is
cleaved by restriction enzymes.cleaved by restriction enzymes.
2. The cleaved vector segments are ligated with a2. The cleaved vector segments are ligated with a
digested DNA fragment to form an artificialdigested DNA fragment to form an artificial
chromosome.chromosome.
3. Yeast cells are transformed to make a large3. Yeast cells are transformed to make a large
number of copies.number of copies.
They are the largest of the cloning vectors, with aThey are the largest of the cloning vectors, with a
cloning limit of 100-1000 kbcloning limit of 100-1000 kb, however they have, however they have
very low efficiency.very low efficiency.
.

21. YEAST/YAC VECTORSYEAST/YAC VECTORS
CEN4CEN4 is the centromere ofis the centromere of
chromosome 4 ofchromosome 4 of YeastYeast. The. The
centromere will segregate thecentromere will segregate the
daughter chromosomes.daughter chromosomes.
ARSARS is autonomously replicatingis autonomously replicating
sequence, its function is as a yeastsequence, its function is as a yeast
origin of replication.origin of replication.
TRP1TRP1 andand URA3URA3 are yeast selectableare yeast selectable
markers, one for each end, to ensuremarkers, one for each end, to ensure
the right reconstituted YACs survivethe right reconstituted YACs survive
in the yeast cells.in the yeast cells.
TELTEL is the telomeric DNA sequence,is the telomeric DNA sequence,
which is extended by the telomerasewhich is extended by the telomerase
enzyme inside theenzyme inside the yeast cell.yeast cell.
SUP4SUP4 is a gene, which is insertionallyis a gene, which is insertionally
inactivated, for a red-white color test,inactivated, for a red-white color test,
like blue-white screening inlike blue-white screening in E. coliE. coli..
Function: YAC vectors can accept genomic DNA fragments of more
than 1 Mb, and hence can be used to clone entire human genes.
B B
S
pYAC3
SnaBI
BamHI
.

22. .

23. SHUTTLE VECTORSSHUTTLE VECTORS
DefinitionDefinition: They are the vectors that can: They are the vectors that can
shuttle between more than one host,shuttle between more than one host,
for example, one isfor example, one is E. coliE. coli and theand the
other is yeast.other is yeast.
Structure and functionStructure and function: Most of the: Most of the
vectors for use in eukaryotic cells arevectors for use in eukaryotic cells are
constructed as shuttle vectors.constructed as shuttle vectors.
• InIn E. coliE. coli::
• This means that they can surviveThis means that they can survive
and have the genes (ori and ampand have the genes (ori and amprr
))
required for replication andrequired for replication and
selection inselection in E. coliE. coli..
• In theIn the desired eukaryotic cellsdesired eukaryotic cells::
• They can also survive in theThey can also survive in the desireddesired
host cellshost cells, and let the target insert, and let the target insert
sequences take effects.sequences take effects.
E.coli
Yeast
.

24. YEAST EPISOMAL PLASMIDSYEAST EPISOMAL PLASMIDS
StructureStructure of YEpsof YEps
aa oriori: for replication in: for replication in E.coliE.coli
aa ampamprr
: for selection in: for selection in E. coliE. coli
aa 22µµ origin: for replication inorigin: for replication in
yestyest
LEU2LEU2: is homologous gene: is homologous gene
and a selectable marker inand a selectable marker in
yeast, involved in leucineyeast, involved in leucine
synthesis.synthesis.
X geneX gene: a shuttle sequence.: a shuttle sequence.
ori
ampr
2µ origin
LEU2X gene
Function of YEps
• It replicates as plasmids
• It integrates into a yeast
chromosome by homologous
recombination.
YEps
.

25. EXPRESSION VECTORSEXPRESSION VECTORS
• In DNA cloning experiments all the genes cloned areIn DNA cloning experiments all the genes cloned are not expressednot expressed fully because offully because of
weak promotersweak promoters in vector DNA.in vector DNA.
• This can be dramatically improved by placing such genesThis can be dramatically improved by placing such genes downstream of strongdownstream of strong
promoters.promoters.
• An additional problem in maximizing expression of cloned genes in E. coli which isAn additional problem in maximizing expression of cloned genes in E. coli which is
frequently encountered with genes from a heterologous source is that the genefrequently encountered with genes from a heterologous source is that the gene carriescarries
no translation start signalno translation start signal which can be efficiently recognized by the E. coli translationwhich can be efficiently recognized by the E. coli translation
system.system.
• This problem may arise for heterologous genes cloned into any host. Thus, evenThis problem may arise for heterologous genes cloned into any host. Thus, even
though the gene can be transcribed from a promoter within the vector, the resultingthough the gene can be transcribed from a promoter within the vector, the resulting
mRNA is poorly translated and little or no protein product will be synthesized.mRNA is poorly translated and little or no protein product will be synthesized.
• In such cases alternative strategies available areIn such cases alternative strategies available are fusing the gene to amino terminalfusing the gene to amino terminal
region of vector gene that is efficiently translated in the hostregion of vector gene that is efficiently translated in the host or coupling the gene to aor coupling the gene to a
DNA fragment carrying both strong promoter and a ribosomal binding site.DNA fragment carrying both strong promoter and a ribosomal binding site.
• Vectors with this additional feature are calledVectors with this additional feature are called expression vectorsexpression vectors..
.

26. .
T7
expressional
vector
E.COLIE.COLI/T7 EXPRESSION/T7 EXPRESSION
VECTORSVECTORS
• Definition ofDefinition of expression vectorsexpression vectors::
Cloned geneCloned gene→→expression vectorexpression vector→→
hosthost→→fusionfusion proteinprotein..
• StructureStructure
• T7 promoterT7 promoter: a strong promoter;: a strong promoter;
• RBSRBS: ribosome binding site;: ribosome binding site;
• ATGATG: translation initiation condon: translation initiation condon
• MCSMCS: Multiple cloning sites: Multiple cloning sites
• TTTT: transcription terminator.: transcription terminator.
• ampamprr
,,.. ori,ori,
• His-tagHis-tag: Some expression vectors are: Some expression vectors are
designed to have six histidine codonsdesigned to have six histidine codons
that encode a hexahistidine tag at thethat encode a hexahistidine tag at the
N terminus of the expressed protein,N terminus of the expressed protein,
which allows one-step purification onwhich allows one-step purification on
an affinity column containing Nian affinity column containing Ni2+2+
..
T7
RBS MCS
TT
ATG

27. INSECT CELL/BACULOVIRUSINSECT CELL/BACULOVIRUS
DefinitionDefinition: Baculovirus is an: Baculovirus is an ♣♣ insect virusinsect virus whichwhich
can be used for the overexpression ofcan be used for the overexpression of ♣♣ animalanimal
proteinsproteins ♣♣ inin insect cell cultureinsect cell culture..
MechanismMechanism::
• Viral promoterViral promoter: This viral gene has an: This viral gene has an extremelyextremely
active promoter.active promoter.
• Insect cell cultureInsect cell culture: The same promoter can be: The same promoter can be
used to drive the over-expression of a foreignused to drive the over-expression of a foreign
gene engineered into the baculovirus genome.gene engineered into the baculovirus genome.
FunctionFunction: This method is being used increasingly: This method is being used increasingly
for large-scale culture of proteins offor large-scale culture of proteins of animalanimal
originorigin, since the insect cells can produce many, since the insect cells can produce many
of the post-translational modifications of animalof the post-translational modifications of animal
proteins, which a bacterial expression systemproteins, which a bacterial expression system
cannotcannot..
Baculovirus-infected SF21 cells.

28. .
MAMMALIAN CELL/VIRAL VECTORSMAMMALIAN CELL/VIRAL VECTORS
• SV40SV40: This virus can infect a: This virus can infect a
number of mammaliannumber of mammalian
species. The SV40 genome isspecies. The SV40 genome is
only 5.2 kb in size.only 5.2 kb in size.
• Since it has packagingSince it has packaging
constraints similar to phageconstraints similar to phage λλ,,
so it can be not used forso it can be not used for
transferringtransferring largelarge fragments.fragments.

29. .
MAMMALIAN CELL/VIRALMAMMALIAN CELL/VIRAL
VECTORSVECTORS
• RetrovirusesRetroviruses: They have a ssRNA: They have a ssRNA
genome, which is copied into dsDNAgenome, which is copied into dsDNA
after infection. The DNA is then stablyafter infection. The DNA is then stably
integrated into the host genome by aintegrated into the host genome by a
transposition mechanism.transposition mechanism.
• They have some strong promoters, andThey have some strong promoters, and
they have been considered as vectorsthey have been considered as vectors
forfor gene therapygene therapy, since the foreign DNA, since the foreign DNA
will be incorporated into thewill be incorporated into the hosthost
genomegenome in ain a stable mannerstable manner..