TERM PAPER

ON
CLONING VECTORS
FOR ANIMALS
SUBJECT:- CONCEPTS IN BIOTECHNOLOGY
SUBJECT CODE:-BTC-012

SUBMITTED TO:- SUBMITTED BY:-

Mr. Harsh Kumar Vipin Kumar Poswal

(Lect. In Biotechnology) B-Tech (Bio Tech) 3rdsem

Roll no- 37, Sec- B

Reg. no.1040070086

ACKNOWLEDGEMENT
I gratefully acknowledge my indebtedness to my respected teacher

MR.HARSH SIR who is always a source of inspiration for me, for

providing this opportunity. Under his able guidance, I have learned

how to overcome the odds and trying circumstances.

I am very thankful to my colleagues with whom

I could discuss and give the final shape to this term paper.

At the same time, I am also thankful to my class incharge

Dr. PRABHOJOT JASSAL who gave us an immense help in the

completion of this project.

INTRODUCTION
 A vector is any vehicle used to transfer foreign genetic material

into another cell. The four major types of vectors are plasmids,

bacteriophages and other viruses, cosmids, and artifical chromosomes.

Common to all engineered vectors are an origin of replication, a

multicloning site, and a selectable marker.

The term paper covers all the topics related to cloning vectors in

animals, including -> History of cloning ,what are vectors ,their

features, plasmids, transcription in vectors, expression vectors ,and

the various types of cloning vectors used in animal cloning. The

various types of cloning vectors which are covered ahead are are SV

40 vectors, BPV vectors, Retrovirus vectors, Polyomavirus vectors,

Vaccinia vectors, P Element vectors, and the Bacculovirus vectors,

benefits of cloning.
BRIEF HISTORY HISTORY OF ANIMAL
CLONING
Cloning was first reported in the early 1950 s. Throughout the 50 s
and 60 s scientists were busy using Nuclear Transfer to clone
amphibians. Most of these experiments went nowhere because the
cloned amphibians were cloned with nuclei from cells that were
actively dividing. This resulted in things like cloned frogs that never
made it passed the tadpole stage. In the 1970 s animal cloning was
first attempted. Mice, sheep and cattle were the prime targets of
70 s genetic cloning, or more like cloning research. By the mid
1980 s scientists had cloned sheep and cattle by using nuclei from
embryos, proving that animal cloning was, at least, possible. In
1995 scientists Campbell and Wilmut created two live lambs. They
were cloned using quiescencial cells. This is when it was discovered
that this kind of cell was best for cloning because it wasn t in any
stage of cell division. Most recently, in 1997, the world famous
Dolly was cloned from an adult ewe s cell. Cloning technology is
moving forward at an accelerated rate. We may even see human
clones in the next twenty years, but we may not.(If only because
of moral issues.)
 VECTORS
In molecular biology, a vector is any vehicle used to transfer foreign
genetic material into another cell. The four major types of vectors are
plasmids, bacteriophages and other viruses, cosmids, and artifical
chromosomes. Common to all engineered vectors are an origin of
replication, a multicloning site, and a selectable marker.

The vector itself is generally a DNA sequence that consists of an insert

(transgene) and a larger sequence that serves as the "backbone" of
the vector. The purpose of a vector which transfers genetic information
to another cell is typically to isolate, multiply, or express the insert in
the target cell. Vectors called expression vectors (expression
constructs) specifically are for the expression of the transgene in the
target cell, and generally have a promoter sequence that drives
expression of the transgene. Simpler vectors called transcription
vectors are only capable of being transcribed but not translated: they
can be replicated in a target cell but not expressed, unlike expression
vectors. Transcription vectors are used to amplify their insert.

Insertion of a vector into the target cell is generally called transfection,

although insertion of a viral vector is often called transduction.

PLASMIDS
Plasmids are double-stranded generally circular DNA sequences that
are capable of automatically replicating in a host cell. Plasmid vectors
minimalistically consist of an origin of replication that allows for semi-
independent replication of the plasmid in the host and also the
transgene insert. Modern plasmids generally have many more
features, notably including a "multiple cloning site" which includes
nucleotide overhangs for insertion of an insert, and multiple restriction
enzyme consensus sites to either side of the insert. In the case of
plasmids utilized as transcription vectors, incubating bacteria with
plasmids generates hundreds or thousands of copies of the vector
within the bacteria in hours, and the vectors can be extracted from the
bacteria, and the multiple cloning site can be restricted by restriction
enzymes to excise the hundredfold or thousandfold amplified insert.
These plasmid transcription vectors characteristically lack crucial
sequences that code for polyadenylation sequences and translation
termination sequences in translated mRNAs, making expression of
transcription vectors impossible.

VIRAL VECTORS

Viral vectors are generally genetically-engineered viruses carrying

modified viral DNA or RNA that has been rendered noninfectious, but
still contain viral promoters and also the transgene, thus allowing for
translation of the transgene through a viral promoter. However,
because viral vectors frequently are lacking infectious sequences, they
require helper viruses or packaging lines for large-scale transfection.
Viral vectors are often designed for permanent incorporation of the
insert into the host genome, and thus leave distinct genetic markers in
the host genome after incorporating the transgene. For example,
retroviruses leave a characteristic retroviral integration pattern after
insertion that is detectable and indicates that the viral vector has
incorporated into the host genome.
 TRANSCRIPTION IN VECTORS
Transcription is a necessary component in all vectors: the premise of a
vector is to multiply the insert (although expression vectors later also
drive the translation of the multiplied insert). Thus, even stable
expression is determined by stable transcription, which generally
depends on promoters in the vector. However, expression vectors
have a variety of expression patterns: constitutive (consistent
expression) or inducible (expression only under certain conditions or
chemicals). This expression is based on different promoter activities,
not post-transcriptional activities. Thus, these two different types of
expression vectors depend on different types of promoters.
Viral promoters are often used for constitutive expression in plasmids
and in viral vectors because they normally reliably force constant
transcription in many cell lines and types.
Inducible expression depends on promoters that respond to the
induction conditions: for example, the murine mammary tumor virus
promoter only initiates transcription after dexamethasone application
and the Drosphilia heat shock promoter only iniates after high
temperatures.

Expression
Expression vectors require not only transcription but translation of the
vector's insert, thus requiring more components than simpler
transcription-only vectors. Expression vectors require sequences that
encode for:

• Polyadenylation tail: Creates a polyadenylation tail at the end of

the transcribed pre-mRNA that protects the mRNA from
 exonucleases and ensures transcriptional and translational
termination: stabilizes mRNA production.
• Minimal UTR length: UTRs contain specific characteristics that
may impede transcription or translation, and thus the shortest
UTRs or none at all are encoded for in optimal expression
vectors.
• Kozak sequence: Vectors should encode for a Kozak sequence in
the mRNA, which assembles the ribosome for translation of the
mRNA.

Features of vectors
Modern vectors may encompass additional features besides the
transgene insert and a backbone:

• Promoter: Necessary component for all vectors: used to drive

transcription of the vector's transgene.
• Genetic markers: Genetic markers for viral vectors allow for
confirmation that the vector has integrated with the host
genomic DNA.
• Antibiotic resistance: Vectors with antibiotic-resistance open
reading frames allow for identification of which cells have
uptaken the vector through antibiotic selection.
• Epitope: Vector contains a sequence for a specific epitope that is
incorporated into the expressed protein. Allows for antibody
identification of cells expressing the vector.
• β-galactosidase: Vector's multiple cloning site contains sequence
for β-galactosidase, an enzyme that digests galactose, to either
side of the region intended for an insert.
• Targeting sequence: Expression vectors may include encoding
for a targeting sequence in the finished protein that directs the
expressed protein to a specific organelle in the cell.
 Different Types of Vectors Used for Gene
Transfers in Animals -
Vector Derived from Features
1. SV40 vectors

(a) Early region Replacement of large-T gene of SV40 1. Produce virions, which infect
replacement vectors host cells
2. Transient gene expression
3. Mammalian cells are hosts
(b) Late region Replacement of VPI, VP2, and VP3 of SV40
replacement vectors genes of SV40, e.g., SVGT-5

(c) Plasmid vectors Origin of replication and large-T gene --

of SV40
(d) Shuttle plasmid Plasmid vector plus pBR322 origin --
vectors and amp' gene, e.g., pSV2, pSV3,
etc.

Rous sarcoma virus promoter in place Strong expression of the

of SV40 early promoter, e.g.,pRSV. marker gene

(e) Passive SV40 transcription regulatory and Shuttle vectors; used for gene
transfecting vectors polyadenylation sequences, plus integration in mammalian cells
pBR322 origin and amp gene

2. BPV vectors Bovine papilloma virus "Transforming Shuttle vectors; often pBR322
region" + pBR322 sequences. sequence deleted prior to
transfection; plasmid-like
vector

3. Retrovirus pBR322 + retrovirus sequences Shuttle vectors; integrates as

vectors provirus into mammalian
genome; produces virions
4. Polyomavirus Polyoma virus origin and early region Similar to SV4O vectors;
vectors + pBR322 sequences mouse cells used as host

5. Vaccinia virus DNA insert placed within the Promising as live

thymidine kinase gene of virus by a
process of recombination.
6. P element vectors Drosophila transposable element P;
minimum of 31 bp inverted repeat
borders and the neighbouring
regions, plus an E. coli vector, e.g..
pUC8; DNA insert of up to 40 kb
placed within the two borders.
vaccines;DNA insert is a
pathogen gene encoding an
antigen
Gene transfer in drosophila; a
helper p element is needed to
provide the transposase
necessary for transposition of
the recombinant p vector into
the drosophila genome

7. Bacculovirus Nuclear polyhedroma virus (NPV) Produce virions;expression

vectors polyhedrin gene replaced by DNA
insert; e.g., AcNPV (Autographa
californica nuclear polyhedroma
virus) and BmNPV (Bombyx mori
nuclear polyhedroma virus) vectors
vector for production of
transgenic proteins in silk
worm larvae (BmNPV vectors)
and in spodoptera frugiperda
larvae.

SV40 Virus –

SV 40 is a spherical virus with double stranded circular DNA of size 5.2

kb. The viral protein contains three viral coded proteins.VP1 is the

major protein present in the capsid with a size of 47000 kDa. Two
more proteins VP2 and VP3 are also present. The DNA of virus is

associated with the four histones (H4,H2A,H2B and H3) proteins. The

viral DNA can be segmented into five precise segments coding for five

different proteins small T, large T, VP1, VP2 and VP3. VP1 coding

region overlaps VP2 and VP3 in a different translation reading frame.

SV 40 virus infects monkey kidney cell lines. The virus travels to the

nucleus and gets uncoated. Then both the T -genes located near the

origin are translated in the clockwise direction. The large T protein is

important for virus DNA replication and starts after the translation of

large T -protein. Replication starts at the origin and is bi-directional. It

terminates when two replication forkmeets. About 105 molecules of

duplex DNA are synthesized per cell. Along with DNA replication, VP1,

VP2 and VP3 proteins are synthesized. Then packing of DNA occurs to

form new virions, which are released by the lysis of cell. The entire

process can also be initiated by transfection with naked SV 40 DNA. SV

40 vectors are constructed similar to phage vectors. Portions of the

viral genome are removed and replaced by other DNA segments.

There are three types of SV 40 vehicles each of which have a distinct

advantage or disadvantage among themselves.

- SV40 Plasmid Vectors –

 - These vectors multiply in the monkey cell line but are not packed

as the virions. These plasmids/ vectors usually contain origin of

replication sequences and larger T-protein gene but do not

contain VP1, VP2 and VP3 genes. They are shuttle vectors, and

have the ability to multiply both in E coli and monkey cell line.

Normally the recombinant is multiplied in E coli cells to high copy

number and then transferred into the cell line. These cells are

stable in bacterial cell and are efficiently transferred from parent

cells to daughter cells. However, the plasmid vectors are unstable

in most animal cells and cannot be maintained indefinite.

Bovine Papilloma Virus Vector –

Bovine papilloma virus (BPV) causes warts (uncontrolled epithelial

proliferation) in cattle. It is a member of a group of viruses which

induces warts and papillomas in a range of mammals. BPV normally

infects terminally differentiated squamous epithelial cells.

BPV has a capsid protein surrounding a circular double-stranded DNA

of size 79 kb. 69% of this genome is important for viral function,

whereas 31 % of the genome can be replaced by the insert.The

recombinant BPV is constructed by ligating the insert and BPV vector

(69%) onto the pBR 322 plasmid, thus generating the shuttle vector

containing plasmid ori site and virus replication sequences.

These shuttle vectors are multiplied in E.coli cells first and then they

are transformed into mouse cell line. It has been observed that if

these sequences are removed prior to transfection, the vector exists at

high copy number i.e., 200 copies per cell.

BOVINE PAPILLOMA VIRUS

When transfected with pBR 322 sequences, it exists at low copy

number, i.e., less than 10 copies cell .The major advantage of BPV is

the generation of permanent cell line. As the infected cells are not

killed, a stable plasmid number is found even when the insert is of

large size.

The selection of transformants is very easy as they form a pile of cells

on the transferred monolayer of cells called "Focus".

RETROVIRUS VECTORS –

Rectroviruses have single stranded RNA genomes which are reverse

transcribed by reverse transcriptase to yield DNA double strand copies
inside the host cells. The DNA copy integrates into the host genome to
become a provirus, which causes permanent transfection of the cells.
The provirus genome is transcribed and expressed; virions are formed
and extruded into the medium.
The following properties of retroviruses are useful in their use as
vectors;
(1) a wide host range (birds,mammals,and other animals),
(2) infected cells are not killed and they continue to produce virus
particles over indefinite period
(3) presence of strong promoters and
(4) regulation of promoter action in case of some
viruses,viz.,murine mammary tumour virus.
Retrovial vectors are constructed from cloned DNA genomes of
retroviruses. These vectors have the following three features;
(1) These vectors have viral sequences for replication, gene expression
(2) DNA inserts may either replace or be located in the nonessential
coding region of the viral genome.
(3) The vector and the recombinant DNAs are packaged into virions
and used as transducing phages.
(4) The viral proteins are usually provided by a helper virus or a
provirus.
(5) DNA copies of the retrovirus genomes are used as vectors,
generally as shuttle vectors.

A typical vector has the following sequences:

(1)It contains pBR322 ori and a selectable marker.

(2) retroviral 5'-LTR and 3'-LTR (long terminal direct repeats),
(3) The vector and the recombinant DNAs are packaged into virions
and used as transducing phages.
(4) The viral proteins are usually provided by a helper virus or a
provirus.
(5) DNA copies of the retrovirus genomes are used as vectors,
generally as shuttle vectors.

BOVINE POLYOMAVIRUS
Polyomaviruses belong to the family polyomaviridae. These viruses
have been referred to as small DNA tumor viruses because of their
ability to transform established cell lines, to immortalize primary cell
cultures and induce tumors in animals. Although potentially oncogenic,
the viruses rarely cause disease in healthy individuals, but in immuno-
compromised humans and animals the consequences can be serious.
Members of the genus include Primate polymavirus SV40, Human
polyomavirus JC and BK, Murine polymavirus, Hamster papovirus and
Bovine polyomavirus (BPyV).

BPyV was originally thought to be of simian origin but it was later

shown to be of bovine origin, the primate cultures having been
contaminated through the use of infected foetal bovine serum. This
was of major significance to the biotechnology industry, as the
presence of BPyV in serum batches poses a serious risk for the
contamination of human therapeutic products.

The virus has been the focus of increasing regulatory concern due to
its reported presence in a high proportion of bovine serum together
with indications that BPyV is zoonotic (naturally transmitted between
vertebrate animals and humans). Recent European guidelines state
that “Serum manufacturers and users are encouraged to apply
infectivity assays for BPyV in order to limit or eliminate infectious virus
from batches of serum.” The FDA is also involved in discussions
regarding the risks associated with polyomaviruses in vaccine
substrates

VACCINIA VIRUS VECTORS

Vaccinia virus is a close relative of the variola virus that causes small

pox. The pox viruses have large upto 300kb dsDNA genomes ,which
replicate in the host cell cytoplasm rather than the nucleus. Therefore

they carry the entire DNA replication and transcription machinery in

their particles and the information for the same in their genomes.

Recombinant viruses are produced by homologous recombination,

using a targeting plasmid transfected into vaccinia virus infected cells.

More recently direct ligation vectors have been developed; these

vectors are transfected into cells containing a helper vaccinia virus to

support replication and transcription of the recombinant

DNA.Recombinant vaccinia DNA is identified using one of the following

strategies;

(1) The transgene is inserted into the viral tk gene and negative

selection,using the thymidine analogue 5-bromodeoxyuridine,is carried

out to minimize the virus type vaccinia genomes.

(2) The transgene is inserted into the viral haemagglutinin locus.The

transfected cells are plated and chicken erythrocytes are added to the

plate . The recombinant plaques remain white , while the wild type

plaques turn red.

(3) Selectable markers ,such as neo, or screenable markers like lacZ

or gusA can be cointegrated with the transgene for direct identification

of therecombinants

P ELEMENT VECTORS
Drosophila P elements have been developed as valuable vectors for

this invaluable genetic material.P elements are transposons of 2.9kb;

they contain 3 genes flanked by short 31bp inverted repeat sequences

at their ends. The genes encode transposase, which recognizes the

terminal repeats and effects transposition of the p elements . A P

Element cloning vector is essentially a bacterial plasmid vectors ,eg.

pUC8, that contains two p elements. One of these P elements has both

its terminal repeats intavt and is able to transposase; the DNA insert is

placed within the transposase.The other P element has its terminal

repeatintact and is able to transposase; the DNA insert is placed within

the transposase gene of this element. P element vectors are shuttle

vectors ; like YACs, they are propagated in Ecoli and are used for gene

cloning in drosophila.After the DNA insert is integrated in the P

element with intact terminal repeats, the plasmid DNA is microinjected

into fruitfly embryos. Transposase is produced by other P element that

lacks the terminal repeats; this transposase directs the transposition

of the DNA insert containing P element into drosophila genome .

BACULOVIRUS VECTORS

Bacculovirus vectors have been developed for transfection of

insects. Two nuclear polyhedrosis viruses (NPV) eg. AcNPV

(Autographa californica NPV), and BmNPV (bombyx mori NPV) have

been exploitedfor this purpose . The NPV polyhedron gene has a very

strong promoter and the polyhedron protein is not needed for npv

replication. Therefore the general strategy is to replace the NPV

polyhedron coding sequence by the DNA insert so that the polyhedron

promoter drives the expression of the transgene. The recombinant NPV

vectors form virions infect silk worm larvae and replicate to yield upto

50 microgram vector DNA per larvae. BmNPV vectors sre used for

infection of silkworm larvae while AcNPV vectors are multiplied and

expressed in the larvae of the insect spodoptera frugiperda.

Retrovirus and transposons vectors integrate into the genome of host

cells in a manner similar to the natural retroviruses and transposons.

Both circular and linear vectors can integrate into the host genome but

the later are far more readily integrated than the former. It has also

been found that the presence of additional vector DNA along with the

integrated gene construct interferes the expression of introduced

gene.Therefore it is often desirable to introduce the transgene with

aminimum of vector DNA associated with it.

BENEFITS OF ANIMAL CLONING

Scientists believe that animal cloning will one day advance agricultural

practices and medicine, and even prevent the extinction of endangered

animals. In agriculture, cloned cattle could produce a higher yield of

meat or milk. The pharmaceutical industry already uses cloned animals

to produce drugs for human use. For example, PPL Therapeutics in

Scotland has generated sheep that produce milk containing a protein

that helps in the treatment of hemophilia. One day pharmaceutical

firms may clone large populations of genetically modified animals to

quickly and inexpensively derive this protein for use in drug products.

Cloned animals could also improve laboratory experiments.

Researchers could create many genetically identical animals to reduce

the variability in a sample population used in experiments, making it

easier for scientists to evaluate disease. Moreover, scientists could

clone a large number of animals that suffer from a human disease,

such as arthritis, to study the disease’s progression and potential

treatments. Some cloned animals such as sheep and pigs live for

years, and scientists could use these animals to evaluate their long-

term response to drug treatments.

R E F E R E N C E
1. Biotechnology (Expanding horizons) by B.D
Singh

2. www.wikipedia.com

3. www.altavista.com

4. Microsoft Encarta encyclopedia.