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Summary Gene Technology, MOB20306, WUR
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A summary of all lectures and scientific articles of the course Gene Technology, MOB20306, at WUR
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Gene technology summaryLecture 1: gene transfer to mammalian cells
To study the function of mammalian genes, we must use three steps:
1. Isolate and clone the gene of interest
2. Manipulate the sequence of the isolated gene
3. Return the altered gene back into eukaryotic cells do determine its function’
Gene transfer is used to identify the regulatory sequences that control gene expression. Transfer of
new or altered genes into new cellular environments provides means to determine gene function.
Gene transfer into mammalian cells is an inefficient process, so an abundant source of starting cells is
necessary to end up with a workable number. Many permanently growing cells lines originate from
tumours. Tumour cells grow quickly and indefinitely in chemically defined media.
Primary cell cultures: normal animal tissues or whole embryos are commonly used to establish this.
The identification and preparation of various protein growth factors that stimulate the replication of
specific cell types, made it possible to grow various types of specialised cells.
Fibroblast: the cell type that usually predominates in a culture. These cells are not defined, but
represent whatever grows when a tissues or an embryo is placed in a culture. Cultured fibroblasts
have the morphology of tissue fibroblasts, but they retain the ability to differentiate into other cell
types. Fibroblasts proliferate longer than other cells and therefore become the predominant cell
type. Even though its lifetime is limited, a single culture can be studied through many generations.
Cell strain: a lineage of cells originating from one primary cell culture.
Cell line: a population of immortal cells (usually do to genetic changes).
Tumour viruses: infect mammalian cells, insert their own genetic material, and hijack the cellular
biosynthetic machinery to manufacture more viruses. Often viral multiplication kills the host cell.
Sometimes, the presence of tumour virus genes permanently alters the growth properties of an
infected cell, transforming it into a tumour cell.
Methods for introducing DNA into mammalian cells:
Calcium-phosphate co- Electroporation Lipofection Viral vectors
precipitation
Cells efficiently take up Cells are placed in a (salt- DNA can be incorporated DNA was isolated from
DNA when it is in the free) solution containing into artificial lipid purified viruses and
form of a precipitate with DNA and subjected to a vesicles, liposomes, that introduced into cultures
calcium phosphate. With brief electrical pulse that fuse with the cell of uninfected cells. The
the calcium phosphate- causes holes to open membrane, delivering ability of virions
coprecipitation method, transiently in their their content directly into (individual viral particles)
purified tumour virus membranes. DNA enters the cytoplasm. Because to introduce their
DNA could transform through the holes directly of the lack of proteins, contents into the
normal cells into cancer into the cytoplasm. plasmid-liposome cytoplasm and nuclei of
cells. complexes are relatively infected cells has been
non-immunogenic. They adapted. New genes are
cannot replicate and can introduced by packaging
carry material of them as virions.
unlimited size.
Restriction enzymes are used to cut tumour virus DNA into fragments.
The basis of classifying viruses is the relation between the viral mRNA and the nucleic acid of the
infectious particle.
, Retroviruses: single stranded RNA viruses with 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. The env gene encodes the envelope glycoprotein that extends from the
lipid membrane of the virion and functions as a ligand for the cellular viral
receptor.
The retroviral envelope fuses directly with the plasma membrane. The
nucleocapsid enters the cytoplasm of the cell, where viral reverse transcriptase
copies the RNA into a dsDNA (double stranded DNA). Provirus: the integrated
viral DNA. The LTR of retroviruses functions as a strong promotor for
transcription of viral DNA. Infected cells can replicate and thereby produce
daughter cells with proviral DNA. Retroviruses thus achieve stable integration.
Eukaryotic helper cells work as retroviruses, however, some genes are deleted so
that the cell cannot produce any viruses. The presence of only the 5’ and the 3’-
LTR and a short sequence, psi, the packaging sequence, is sufficient to confer
packaging.
Adenoviruses: non-enveloped, linear, double-stranded DNA viruses. Adenoviral genes are grouped as
early (E) and late (L) genes. 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. Advantages: most humans are susceptible to adenoviruses, they can deliver transgenes to
(non)dividing cells, are stable and resistant to physical manipulations and the life cycle does not
require integration into the host cell genome. Disadvantages: the induction of a significant host
immune response (local inflammatory reaction, which can result in cell lysis) and the development of
virus-neutralizing antibodies.
Retroviral vectors have a low cloning capacity. Also, retroviral vectors must be
integrated into dividing cells. This often happens ex vivo: cells are removed from the
body, treated to stimulate replication and then transduced with the retroviral vector
before being returned to the patient.
Microinjection of cloned genes into fertilised eggs (see image): mainly used when
producing transgenic animals. The highest efficiency of this procedure is achieved with
mice. The percentage of eggs that survive is usually between the 10 and 30 percent. Of
the survivors, the number that have the foreign DNA integrated into their
chromosomes is between a few percent and 40 percent. Transgenic mice: they carry
the foreign gene. The foreign DNA is the transgene. This procedure is mainly used to
over-express a gene of interest (a gain of function model). Direct injection gives no
opportunity to manipulate or control DNA integration and expression. This can be
achieved by using lipofection or electroporation into special cells, embryonic stem cells
(ES cells).
One of the basic principles of recombinant DNA technology is the use of biological
markers to identify cells carrying recombinant DNA molecules. In bacteria, these are
commonly antibiotic-resistance genes. The enzyme aminoglycoside
phosphotransferase (APH) inactivates the neomycin related drug G418, which kills mammalian cells
by blocking protein synthesis. This gene can be added (under control of an eukaryotic promoter) to
the vector carrying the gene of interest to be integrated into the target host genome. In case of using
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