College aantekeningen
Lecture notes Gene Technology, MOB20306, WUR
- Instelling
- Wageningen University (WUR)
All lectures are noted in this document. From the course Gene Technology, MOB20306 at WUR
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Gene technology lecture notesLecture 1: Gene transfer to mammalian cells and gene therapy
Gene therapy involves reversed genetics. With forward
genetics, you want to discover what gene is responsible for a
phenotype. Reversed genetics is the other way around: you
know the gene and you want to alter it/knocking it out to
obtain a certain phenotype (see image).
There are three steps involved with gene transfer to
mammalian cells:
1. Clone the gene
2. Manipulate the gene in vitro
3. Return the gene into an organisms or single cells (this
is the most difficult part).
Why do you want to perform gene transfer? To treat diseases (gene therapy), to produce certain
molecules/proteins (during biotechnology/pharmaceutics) and to track the function of certain
proteins (study gene function).
Return the genes into single cells: the first step is to obtain the cells by starting a cell line. The first
step for this is to isolate individual cells from a tissue by disrupting the extracellular matrix and cell-
junctions. Mammalian cells require a solid surface that is coated with material they can adhere to
(this property can be used to obtain specific cell
types based on surface properties and their binding
to antibodies). A FACS (fluorescence activated cell
sorter) is used to detect fluorescence. Antibodies
coupled to a fluorescent dye can be labelled to find
specific cells. Droplets containing single cells are
given a positive/negative charge depending whether
the cell is fluorescent. They are collected into
collection tubes based on their charge.
Another way to collect single cells is by using a laser
(see bottom image). Here, the laser can cut a certain
labelled piece out of a tissue and a second laser can
collect these cells.
Primary cell cultures: cell cultures directly prepared
from tissues of an organism. Repeated culturing can
result in a cell line (which are immortalised by
mutations). Cell lines can be most easily be generated
from cancer cells, because these cells are already
mutated/immortalised. Growth factors are needed to
stimulate replication of specific cell types. Cells in a
culture remain differentiated. Plant cell lines can be
made from plant hormones, animal cell lines from
tumours.
,Transformation methods for animal cells:
Calcium-phosphate co-precipitation Electroporation
Traditionally done using adenovirus DNA.
Stable expression means integration in the genome
so that the DNA persists in all cells derived from the
initial few cells that were transformed.
Lipofection Viral vectors
To reproduce, a virus must enter the host, replicate,
transcribe and translate to form new coat proteins,
assemble and escape from the hosts. Coat proteins
are necessary to survive outside of the host.
, Viruses can efficiently infect cells by membrane
fusion, pore formation and membrane
disruption. Non-enveloped viruses usually leave
an infected cell by lysing it, enveloped viruses
(with coat proteins) can leave the infected cell
using
budding.
Adeno-
associated
viruses form circular and linear
episomes and integrate with low
frequency. Human
immunodeficiency viruses (HIV)
integrate with high efficiency.
A retrovirus is enveloped and is a RNA-virus. The life-cycle of a
retrovirus can be seen in the image.
With retroviruses, the long terminal repeats are needed for integration. Once these are in the
genome, it is safe and cannot be packaged into virus particles anymore (the virus cannot replicate
anymore). It can, however, still transcribe and be translated.
The retroviral vector is called pLNCX in the image. The vector can be cloned into E. coli, because this
is an easy organism to use for cloning.
Disadvantages of retroviruses: it has a low cloning capacity, eukaryotic cells should divide to enable
integration (viral DNA cannot pass the nuclear envelope, exceptions for this are the lentiviruses,
which can infect non-dividing cells and integrate DNA into the host genome) and retroviruses cannot
control the integration site in the host genome. A disadvantage of long terminal repeats is that they
have a promotor function and can thus promote the growth of a
new gene. Alternatives for this are the development of self-
inactivating vectors or the development of non-integrated retro-
lentiviruses.
Adeno-associated viruses: single strand DNA virus, replication
defective (needs an Adenovirus/Herpes simplex virus), has an
apparent lack of pathogenicity, can infect (non)dividing cells and
does not generally integrate into the genome. ssDNA genome is
packaged within a non-enveloped icosahedral capsid. The ssDNA contains 3 ORFs (rep, cap & AAP)
flanked by inverted terminal repeats (ITRs). rep encodes four non-structural proteins (Rep40, Rep52,
Rep68 and Rep78) essential for replication, transcriptional regulation, and virus particle assembly and
cap encodes 3 structural proteins (VP1, VP2 and VP3) that form the 60-mer viral capsid with the aid
of the assembly-activating protein (AAP). To generate a recombinant adeno-associated virus, a gene
of interest is inserted between the ITRs to replace the rep and cap proteins.
There are between 5000 and 8000 known diseases caused by a single gene defect. Gene therapy can
be carried out in three routes:
1. Gene addition (most basic example is the micro-injection in a pronucleus of a fertilized egg).
Advantages: highly efficient with mice, transgenic progeny is heterozygous (not chimeric*)
for transgene and no selection marker is needed.
Disadvantages: random integration of DNA in the genome and
low efficiencies in other species than rodents. Not for humans.
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