EXPRESSION OF CLONED GENES IN ANIMAL

Introduction:

Animal cells are advantageous for the production of recombinant animal protein
because they perform desired post- translational modification that are not carried out by
bacterial cells and hence used on a commercial scale to synthesize product such as
antibodies, hormone, growth factors, cytokines and viral coat protein for immunization.
There are several reasons for attempting to express cloned gene, including
 The production of large quantities of labeled cRNA to be used as probes.
 The construction of expression libraries (cDNA libraries where the cloned
DNA of each clone is expressed, allowing screening for structural or
functional properties of the encoded polypeptide)
 the analysis of gene function at the protein level
 the commercial production of proteins
 the production of antibodies
 the entrapment of interacting molecules.
A problem with any expression strategy is the stability of the expression vector.
Cells expression large amounts of a foreign protein (or even an endogenous protein) grow
more slowly than wild type cells, hence there is selection of mutants which have lost the
expression vector altogether or have reduced expression levels. The use of inducible
promoters can help to avoid this problem as large quantities of cells containing the vector
can be grown, then expression can be induced and the cells harvested quickly. Other
important aspects of expression strategy include the control of vector segregation and the
design of expression vectors to limit the likelihood of spontaneous occurring structural
mutation.
The addition of a leader peptide sequence to the vector allows the expressed
protein to be secreted from the cell. This may increase stability by removing over
expressed protein from the intra cellular environment and the may be necessary for cell
survival if the expressed protein is toxic. In any case, secretion facilitates the purification

1 Prof. Dinesh Yadav, Department of Biotechnology, DDU Gorakhpur

University. E-mail:dinesh_yad@rediffmail.com
of the protein from cell culture. A vector containing a leader peptide upstream of the
expression cloning site is a secretion vector.
Eukaryote expression hosts
Where bacterial cell fail to process expressed protein correctly, eukaryote cells
may be used as alternative expression hosts. Expression protein in eukaryotic cells also
allows the analysis of protein function in a eukaryotic environment, and use of
intracellular signal sequence allows protein targeting to particular organelles.
Three type of eukaryotic host are used for protein over expression.
a. Yeast
b. Insect
c. Mammalian cells.
Mammalian cells provide the ultimate expression systems for human therapeutic
protein. Few mammalian expression vectors are maintained episomally, so long-term
expression requires constructs that integrate into the genome. Mammalian & expression
construct may be based on bacterial plasmids, which are transected into cells, or
mammalian viruses, which transducer DNA into the cells. Most viral vectors are
recombinant integrating viruses, although vectors containing a herpes virus origin of
replication are maintained episomally. Efficient mammalian expression vectors carry a
strong, constitutive viral promoter (e.g. the SV40 early promoter and enhancer) or an
indocile promoter (e.g. the heat shock promoter or E coli lac promoter). Heterologous
inducible promoters are advantageous because only the foreign gene is induced and not
other endogenous mammalian gene. Expression vector also carry a strong translation
initiation sequence (kozak consensus), a polyadenylation site, and usually an intron,
which may be essential for the efficient expression of some genes. Some of the most
efficient mammalian expression vector exports gene amplification. Certain genes are
maintained at a high number. Analysis of the amplified region (amplicons has shown that
they contain much more DNA then the amplified gene itself. i.e. nonselected DNA is co-
amplified.
Analysis of gene regulation
Reporter gene: Analogous to the principle of expressing cloned gene by sub cloning
them in vector with suitable regulatory elements, cloned regulatory elements can be

2 Prof. Dinesh Yadav, Department of Biotechnology, DDU Gorakhpur

University. E-mail:dinesh_yad@rediffmail.com
“expressed” by subcloning them in vector providing a suitable gene to regulate,
facilitating an essay for gene regulation. In principal any gene will suffice, but it is
convenient to use a gene whose expression can be determined easily and quantitatively
using a simple assay, and whose product is absent from the host cell.
Alternatively reporter vectors are available for the analysis of different regulatory
element:
(i) Promoter probe: Vectors containing a reporter gene downstream of a
polylinker, for the insertion and testing of putative promoter elements.
(ii) Enhancer probe: Vector contain a reporter gene driven by a minimal promoter
and a polylinker for the insertion of putative enhancer elements.
(iii) Terminator probe: Vector for the analysis of bacterial transcription
terminators
The analysis of gene regulation may involve comparing the activity a series of
reporter constructs (reporter gene with upstream cloned regulatory elements), in which
the regulatory elements have been mortified by in- vitro transcription using different cell
lysates, by transient transfection of reporter constrict into cells, or for multicellular
organism, by introducing the construct into the germ line (a reporter transgene). The in-
vitro transcription and cell line approaches are simple experiment but restricted in the
information they provide.
A particular disadvantage of the cell line approach is that the transiently episomal
vector does not accurately represent in-vivo of control afforded by chromatin structure of
distant as regulatory element. Additionally the high copy number of the vector may titrate
out transcription factor.
Reporter transgenic provides amore accurate representation of endogenous gene
regulation and allows the spatial of temporal effect of modulating regulatory element to
be addressed. However, the reporter transgenic maybe subject to position of dosage
effect, resulting in entopic or restricted expression patters of variable expression levels.

Overview of gene transfer strategies

Gene transfer to animal cells can be achieved essentially via three routes.

3 Prof. Dinesh Yadav, Department of Biotechnology, DDU Gorakhpur

University. E-mail:dinesh_yad@rediffmail.com
(i) Direct DNA transfer: this is the physical introduction of foreign DNA directly into the
cell. In culture cells this can be done by microinjection whereas for cells in- vivo direct
transfer is often achieved by bombardment with tiny- DNA coated metal particle.
(ii) Transfection: this encompasses a number of techniques, some chemical and some
physical, which can be used to persuade cells to take up DNA from their surrounding.
(iii) Transduction: the third is to package the DNA inside an animal virus, since viruses
have evolved mechanism to naturally infect cell and introduce their own nucleic acid.
The transfer of foreign DNA into cells by this route is termed transduction.

Major expression system used in animal cells system host major application

System Host Major application

Non replicating plasmid
Vector
Non selection Many cell line Transient assays

Dominant selection marker Many cell line stable transformation,

Long term expression

DHFR/methotrexate CHO cell Stable selection,high,

High level expression

Plasmid with viral replicon

SV 40 replicon CHO cell High level transient
Expression
BPV replicon Various murin Stable transformation
EBV replicon Various murin Stable transformation

Viral transduction

4 Prof. Dinesh Yadav, Department of Biotechnology, DDU Gorakhpur

University. E-mail:dinesh_yad@rediffmail.com
Vector

Adenovirus E1 replacement 293 cell Transient expression

Adenovirous amplicon Various mammalian In vivo transfer
Baculo virus Insect High level transient
Expression
Vaccine virus Mammilion cell High level transient
Expression
Lenti virus Non dividing cell stable transformation

Construct design for high level trans gene expression in animal cells:
Following consideration should apply when high level expression is required.
(i) The use of a strong and constitutive promoter: Very active promoter provides
the highest level of gene expression. The element most commonly used in
mammalian cells are the SV 40 early promoter and enhancer, the Rous
sarcoma virus long terminal repeat promoter and enhance & human
cytomegalo virus immediate early promoter.
(ii) The inclusion of an intron:the presence of an intron in a eukaryotic expression
unit usually enhances expression. The presence of an intron is very important
in construct that are to be expressed in transgenic animal. Most mammalian
expression vectors in current use in corporate a heterogonous intron, such as
the SV– 40 small t-antigene intron or the human growth hormone intron or
modifier hybrid intron that match. The consensus splice donor and acceptor
site sequence.
(iii)The inclusion of a polyadenylation signal: Polyadenylation signal (terminators)
is required in eukaryotic gene to generate a defined 3’ end to the mRNA. This
tail is required for the export of mRNA into the cytoplasm, and also increases
its stability

(iv) The removal of unnecessary untranslated sequences: 5’ and 3’ URRs may be

rich in secondary structure, which presents efficient translation. In animal

5 Prof. Dinesh Yadav, Department of Biotechnology, DDU Gorakhpur

University. E-mail:dinesh_yad@rediffmail.com
 system, UTR sequence is generally removed from transgenic constructs to
maximize expression.
(v) Optimization of the transgenic for translational efficiency: In mammalian
gene translation efficiency increases due to presence of kozak sequence 5’-
CCRCCUAUGG – 3’
(vi) The incorporation of a targeting signal: 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 secretary pathway using a signal peptide. Many
mammalian expression vectors are available for this purpose, and they
incorporate heterologous signal peptide.

Map of some commonly used vectors in mammalian cell:

Shuttle vector conferring selective advantage to mammalian cell lines grow in the
presence of methotrexate. It permits selection in dhfr- cell lines.

6 Prof. Dinesh Yadav, Department of Biotechnology, DDU Gorakhpur

University. E-mail:dinesh_yad@rediffmail.com
Expresses chloramphenicol acetyltransferase in eukaryotic cell lines under the control of
the simian virus 40 early promoter.

7 Prof. Dinesh Yadav, Department of Biotechnology, DDU Gorakhpur

University. E-mail:dinesh_yad@rediffmail.com
Expression vector, maintained at high copy number

8 Prof. Dinesh Yadav, Department of Biotechnology, DDU Gorakhpur

University. E-mail:dinesh_yad@rediffmail.com