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Gene technology summary
- Instelling
- Wageningen University (WUR)
This is a summary of the course gene technology, including figures used in the lectures.
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Voorbeeld 4 van de 31 pagina's
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Gene technologyLecture 1. Gene transfer to mammalian cells
Three essential tools form the basis for studying the function of mammalian genes.
- Isolate and clone the gene of interest
- Manipulate the sequence of the isolated gene
- Return the altered gene back into eukaryotic cells to determine its function
(Im)mortal mammalian cell lines
Many permanently growing cell lines originate from tumors; cells that have escaped from normal
limits to growth. Normal animal tissues or whole embryos commonly are used to establish primary
cell cultures. To prepare tissue cells for culture, trypsin or another protease is used to destroy the
proteins in the junctions that normally interconnect cells. The identification and preparation of
various protein growth factors that stimulate the replication of specific cell types, as well as other
recent modifications in culture methods, now permit researches to grow various types of specialised
cells.
The cell type that usually predominates in cultures is called a fibroblast, because it secretes the types
of proteins associated with fibroblasts in fibrous connective tissue of animals. Cultured fibroblasts
have the morphology of tissue fibroblasts, but they retain the ability to differentiate into other cell
types. Fibroblasts cells, although also destined to die, proliferate for a while and soon become the
predominant cell type. Even though its lifetime is limited, a single culture can be studied through
many generations. Such a lineage of cells originating from one initial primary culture is called a cell
strain. Occasionally, cells undergo some genetic changes allowing them to grow indefinitely. Such
population of immortal cells is called a cell line.
Methods for introducing DNA into mammalian cells
Calcium-phosphate co-precipitation
Cells efficiently take up DNA when it is in the form of
a precipitate with calcium phosphate. It was by using
the calcium phosphate-coprecipitation method that
purified tumor virus DNA could transform normal
cells into cancer cells, a compelling demonstration
that cancerous growth can be encoded in DNA.
Restriction enzymes were used to cut tumor virus
DNA into fragments, which could be tested
individually by transfection, allowing the localisation
of functional genes to specific DNA fragments.
Electroporation
With electroporation, DNA is introduced into bacterial, yeast, plant and animal cells. Cells are mixed
with the DNA to be transfected and placed in a small chamber with electrodes connected to a
specialised power supply. A brief electric pulse is discharged across the electrodes, which transiently
opens holes in cell membranes, bypassing the endocytotic vesicles. DNA enters the cells, which are
subsequently plated on fresh medium. The cultures can be harvested for experiments during the
transient expression phase, or selection can be applied to isolate stably transfected clones.
,Lipofection
DNA can be incorporated into artificial lipid vesicles, called liposomes, that fuse with the cell
membrane, delivering their content directly into the cytoplasm. Plasmid-liposome complexes have
many advantages as gene transfer vectors. Because of the lack of proteins, they are relatively non-
immunogenic. They can carry exogenous material of essentially unlimited size. They cannot replicate
or recombine to form infection agents. Their disadvantage is their relatively low transduction
efficiency as compared to viral vectors.
DNA, which is negatively charged at near-neutral pH due to its phosphodiester backbone, is mixed
with lipid molecules with positively charged (cationic) head groups. The lipid molecules form a bilayer
around the DNA molecules, which creates liposomes that are mixed with cells. Most mammalian cells
are negatively charged at their surface, so the positively charged liposomes interact with the cells.
Cells take up the lipid-DNA complexes, and some of the transfected DNA enters the nucleus.
Viral vectors
Knowledge about mechanisms of viral replication has allowed modification for both DNA- and RNA-
viruses for various purposes. For instance, the ability of virions (individual viral particles) to introduce
their contents into the cytoplasm and nuclei of infected cells has been adapted for use in DNA
cloning and offers possibilities in the treatment of certain diseases. The introduction of new genes
into mammalian cells by packaging them into virions is called viral gene transduction, and the virions
used for this purpose are called viral vectors.
The relation between the viral mRNA and the nucleic acid of the infectious particle is the basis of a
simple means of classifying viruses. In this system, a viral mRNA is designated as a plus strand and its
complementary sequence, which cannot function as an mRNA, is a minus strand. A strand of DNA
complementary to a viral mRNA is also a minus strand. Production of a plus strand of mRNA requires
a minus strand of RNA or DNA be used as a template.
,LTR Retroviruses
LTR Retroviruses are single-stranded RNA viruses that consist of the 5′ and 3′ long-terminal repeats
(LTRs) and the gag, pol and env genes.
- gag: encodes three proteins that form the shell of the virion
- pol: encodes reverse transcriptase, integrase, and ribonuclease H (RNaseH), which are
necessary for viral integration into host chromosomal DNA
- env: encodes the envelope glycoprotein that extends from the lipid membrane of the virion
and functions as a ligand for the cellular viral receptor
Upon interaction with specific host-cell membrane proteins the retroviral envelope fuses directly
with the plasma membrane without first undergoing endocytosis. Following fusion, the nucleocapsid
enters the cytoplasm of the cell; then deoxynucleoside triphosphates from the cytosol enter the
nucleocapsid, where viral reverse transcriptase and other proteins copy the single strand RNA
genome of the virus into a dsDNA copy by a rather complex mechanism.
The gene is cloned into a retroviral vector
that lacks most viral genes. The gene is
usually expressed under the control of the
strong viral promoter in the LTR. The
recombinant (dsDNA) plasmid is transfected
into a special packaging cell line that harbors
an integrated provirus. The provirus has been
crippled so that, although it produces all the
proteins required to assemble infectious
viruses, its own RNA cannot be packaged into
virus. 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 the
foreign gene product.
The viral DNA copy is transported into the
nucleus of the host cell and integrated into
one of many possible sites in the
chromosomal DNA. The integrated viral DNA,
referred to as a provirus, is transcribed by the
host-cell RNA polymerase, generating mRNAs
and genomic RNA molecules. Thereby the LTR
functions as a strong viral promoter. The
host-cell machinery translates the viral
mRNAs into glycoproteins and nucleocapsid
proteins. The latter assembles with genomic
RNA to form progeny nucleocapsids, which
interact with the membrane-bound viral glycoproteins. Eventually the host-cell membrane buds out
and progeny virions are pinched off.
, Because most retroviruses do not kill their host-cells, infected cells can replicate, producing daughter
cells with integrated proviral DNA. These daughter cells continue to transcribe the proviral DNA and
bud progeny viruses. Retroviruses integrate into target cell DNA and thus achieve stable integration
of the transgene. Theoretically, stable integration should provide long-term expression, although
methylation of the LTR often results in downregulation of transgene transcription. Viruses deleted of
one or more of their structural genes are fully infectious when propagated in a Eukaryotic Helper Cell
that expresses the missing genes. When a transgene is inserted into a virus with deleted genes and
that virus infects a target cell, viral sequences containing the transgene integrate into the host
genome and subsequently can stably express the transgene. To this end retroviral vectors have been
developed. Such retroviral vector however, is incapable of undergoing viral replication because the
deleted structural genes that are necessary for this process are not expressed by the target cell. As
such, the deleted viral vector transfers and expresses the transgene, but is incapable of establishing
an active infection in the host. Studies have shown that solely the presence of the 5′ and 3′ LTRs and
a short sequence named psi (ψ) -the packaging sequence- is sufficient to confer packaging when all
of the structural genes are expressed by the helper cell. Transfection of these helper cell lines with a
minimal vector containing the desired transgene results in the production of a defective recombinant
retrovirus. The resulting stock of recombinant retroviruses is termed helper-free, because it lacks
wild-type replication-competent retroviruses. The recombinant retrovirus stock can be used to infect
a target cell culture. The recombinant viral genome is efficiently introduced, reverse-transcribed into
DNA (by reverse transcriptase deposited in the virus by the packaging cells, and integrated into the
genome the target cells. These cells now express the newly virally introduced gene, but they never
produce any virus, because the recombinant retrovirus genome lacks the necessary viral genes. In
other words, the recombinant virus carrying the foreign gene can infect various types of cells but is
replication-deficient because of the missing genes from the virus. The transduction of target cells
with recombinant retroviruses that are defective in viral gene expression also minimizes the
development of an antiviral immune response.
Adenoviruses
Adenoviruses are non-enveloped, linear, double-stranded DNA viruses. Approximately 50 serotypes
of adenovirus have been identified and grouped from A to F on the basis of genome size,
composition, homology, and organization. The most studied adenoviruses belong to single group;
namely group C. The prototype adenoviral vector for gene therapy is also based on this group.
The adenoviral genome is approximately 36 kb. Each end of the viral genome is flanked by 100 to 150
bp of repeated DNA sequence (inverted terminal repeat). Viral gene expression occurs in a sequential
cascade. Adenoviral genes are grouped as early (E) genes whose expression precedes viral DNA
replication and the transcription of late (L) genes at 6 to 8 h after infection. The E genes encode
regulatory proteins for viral replication and the L genes encode structural proteins necessary for
assembly of progeny virions. Adenoviral vectors have been based on the observation that deletion of
some of the E genes results in replication-deficient viruses. Such viruses can be propagated in helper
cell lines that express the E gene products. For example, a helper cell line was prepared from human
embryonic cells stably transformed with Adenovirus DNA fragments that constitutively express the E
proteins. The amount of DNA that can be effectively packaged into wild type adenovirus virions is
~105% of the wild-type genome and thus allows for the insertion of ~2 kb of foreign DNA. With the
viruses with a single deleted E gene (E1), it has been possible to insert 5 to 6 kb of DNA. An additional
2 kb of DNA can be inserted by deletion of the E3 gene. The E3 gene product functions in abrogating
the host immune response to the virus by preventing cytolysis mediated through cytotoxic T cells and
tumor necrosis factor. E3 is not required for viral growth in tissue culture, and E3-deleted viruses
appear to be fully infectious. Adenoviral vectors have a number of potential advantages over
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