Mammalian expression vector

Colorless X gal is cleaved by

beta Galactosidase to produce
Plasmid Vector an insoluble blue product.
For expression in bacteria

A typical mammalian expression vector for high level

protein expression. The vector is stiched together
from several distinct units/ different components of a
functional gene. It carries a potent promoter from
cytomegalo virus (CMV), an intron from betaglobin
gene, and the poly A addition signal from SV 40 virus.
These plasmids carry also several restriction sites
(polylinker). The plasmid backbone carries the usual
sequences for replication (ColE1 ori) and drug
resistance, AmpR. , and an M13 origin of replication in
order to be able to recover the vector as a single
stranded phages for easy mutagenesis
 Chromosomes /
Organism
Haploid Cell Yeast, Saccharomyces cerevisiae, is a eukaryote with 34
(2n) chromosomes;

it reproduces sexually as well as asexually by budding.

In suspension cultures, it grows as single cells with cell

doubling time of 1.5 to 2.5 hrs, but on agar plate cells
produce colonies.

Its haploid DNA content (1.4 x 107 bp) is only 3 times that of
E. coli, and its genetics has been extensively studied.

Yeast viruses are not known.

Only a single yeast plasmid has been discovered, which

has been used to construct some useful vectors.

The tough polysaccharide wall of yeast is an effective

barrier to DNA molecules.

Therefore, yeast cell wall is enzymatically digested to

produce spheroplasts, which can take up DNA following
treatment with CaCl2; walls regenerate in specific media.
Cloning of yeast biosynthetic genes by complementation
in E. coli.

The figure shows a strategy used to clone yeast LEU2

gene. A plasmid library was constructed from yeast
chromosomal DNA using conventional procedures.

The library was transformed into an E.coli leuB mutant

which is deficient in the enzyme beta-isopropylmalate
dehydrogenase that plays a role in the leucine
biosynthesis and coded by LEU2 gene.

Bacteria that take up a plasmid carrying LEU2 gene are

able to express at it at levels sufficient to allow them to
grow in the absence of leucine . These bacteria can be
isolated simply by plating them on leucine-free
plates/media . Plasmids are recovered from the cells.
Shuttle vectors replicate in both E. coli and yeast. This a breakthrough that
advanced our ability to apply recombinant DNA technology to yeast was the
construction of plasmid vectors that can replicate in both yeast and
E.coli.Shuttle vectors contain selectable markers and origins of DNA
replication that work in each organism. Selectable markers like Amp resistance
gene combined with a yeast marker gene like URA3 or LEU2.

Integrating plasmid: Lack sequences for the initiation of replication. . The only
way for them is to stably express them is by directly integrating the plasmid
into yeast chromosome .

Replicating Vectors: exist as free plasmid circles. One of these use replication
origin of from a natural yeast plasmid, the 2m circle.
Others use cellular replicating sequences called Autonomously Replicating
Sequences (ARS) .

ARS containing elements are stabilized by centromere sequences (CEN). This

confers a stable partitioning of the plasmid at cell division and keeps the
number 1-2 per cell
Simplest yeast vector is an
integrating plasmid. It carries
bacterial plasmid sequences from
pUC118 that provide a replication
origin and drug resistance gene for
growth in E.coli, a yeast marker
gene (here LEU2) for selection of
transformants in yeast, and
restriction sites for inserting new
sequences. Yeast stably acquire
this plasmid by integrating it directly
into a chromosome. Linearising the
plasmid before transformation
directs integration to a particular
location.
Replicating vectors exist as free
plasmids circles in yeast. One of the
replicating plasmids uses a replication
origin (ori) from a natural yeast
plasmid, the 2m circle. Others use
cellular replication origins called
autonomously replicating sequences
(ARS) elements. ARS containing
vectors are stabilized by addition of
centromere (CEN) sequence which
confers stable partitioning of the
plasmid at cell division. A centromere
(CEN) sequence also holds a plasmid
copy number to 1- 2 per cell.

Yeast artificial chromosome (YACs) contain both a centromere and two

telomeres which function as stable chromosome ends, allowing YACs to
replicate as small linear chromosomes. YACs can carry several hundred
thousand base pairs of DNA, making them appropriate for specialized
genome mapping procedures
URA3 gene will be killed in the presence of
5FOA (negative selection)
Loss of ODCase (Orotidine 5 phosphate decarboxylase) activity leads to a lack of cell growth
unless uracil or uridine is added to the media.

URA3- Yeast cannot grow unless uracil or uridine is added

The presence of the URA3 gene in yeast restores ODCase activity, facilitating growth on
media not supplemented with uracil or uridine, thereby allowing selection for yeast carrying the
URA3+ . URA3+ yeast grows on media not supplemented with uracil,

In contrast, if 5-FOA (5-Fluoroorotic acid) is added to the media, the active ODCase will
convert 5-FOA into the toxic compound (a suicide inhibitor) 5-fluorouracil causing cell death,
which allows for selection against yeast carrying the gene.
URA3+ yeast dies in the presence of 5 FOA

Since URA3 allows for both positive and negative selection, it has been developed as a
genetic marker for DNA transformations and other genetic techniques in bacteria and many
fungal species. It is one of the most important genetic markers in yeast genetic modification.
While URA3 is a powerful selectable marker it has a high background. This background is
because cells that pick up mutations in URA3 may also grow on 5-FOA. Colonies should be
verified by a second assay such as PCR to confirm the desired strain has been created. Cells
of leu2 strains will be able to grow on the minimal medium (medium lacking leucine) only
when they will be transformed by YEpl3.
Dextran-inert carbohydrate polymer
-Coupled to DEAE (diethyl aminoethyl)
+ vely charged combine with
negatively charged DNA

Microinjection Suction pipette

transfer DNA directly into frog eggs
Suspension cell cultures
Methotrexate inhibits DHFR
 Retrovirus Vectors Provide High Efficiency Vectors for Stable Gene Transfer
1. Most of the viruses like baculovirus, vaccinia virus and SV40 virus etc., are lytic
viruses--- they enter cells, take over the host machinery , replicate massively and
come out as killing the cell in the process . So these vectors can not be used to
introduce a gene stably. This is performed by retroviruses.

2. Retroviruses are RNA viruses with a life cycle different from that of the lytic viruses.
Their RNA genome, upon infection, is converted to a DNA by reverse transcriptase,
a viral enzyme. . Viral DNA is efficiently integrated into the cells chromosomal DNA,
where it resides permanantly.

3. The integrated provirus produces viral RNA from a strong promoter located at the
end of viral genome called LTR.

4. The viral RNA codes proteins as well as becomes genomic RNA for new viruses.

5. Viruses are assembled in the cytoplasm and bud from the cell membrane , usally
without any effect on the cells health.
6. Hence the retrovirus genome becomes a permanent part of the host cell indefinitely
 Design and use of retroviral vectors

Gene is cloned into retrovirus vector for stable,

long-term expression.

Retroviral vector lacks most of the viral genes .

Foreign gene is usally expressed under the control
of the strong viral promoter in the LTR.

Recombinant plasmid is transfected into special

packaging cell linethat harbors integrated provirus.

The provirus is crippled so that it produces all

proteins required for to assemble infectious
particles but it cannot pakage its own RNA.
Instead RNA produced from the recombinant virus
is packaged .
The virus stock released from the packaging cells
thus contains only recombinant virus.

The virus can be used to infect virtually any other

cell type., resulting in the integration of the viral
genome and the stable production of foreign gene
product.