Starting and maintaining a cell line: normal animal tissues or whole embryos are used to establish
primary cell cultures. A protease is used to destroy the proteins in the junctions that interconnect
cells.
Fluorescence-activated cell sorter: in a FACS fluorescence activated cell sorter, an antibody is
coupled to a fluorescent dye to label specific cells. Droplets containing single cells are given a
negative or positive charge, depending on whether the cell is fluorescent. The droplets are deflected
by an electric field into collection tubes according to their charge.
Laser micro-dissection microscope: a method for isolating specific cells of interest from microscopic
regions of tissue or cells. Micro-dissection from tissue slices make use of thin sections of tissues. A
laser beam cuts around the region of interest and a second laser beam used to catapult selected
region into container.
Transformation methods for animal cells
- Ca2+ phosphate co-precipitation: cells efficiently take up DNA when it is in the form of a
precipitate with calcium phosphate. The DNA of adenoviruses is put in a phosphate buffer
and calcium chloride is added. A precipitation is formed. The DNA is added to cells and grown
on a agar plate. The medium is transferred and the cells have taken up the DNA.
- Electroporation: DNA is introduced into 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 opens holes in cell membranes,
bypassing the endocytotic vesicles. DNA enters the cells, which are plated on fresh medium.
- Lipofection: DNA can be incorporated into artificial lipid vesicles, that fuse with the cell
membrane, delivering their content directly into the cytoplasm. DNA (negatively charged) is
mixed with lipid molecules with positively charged head groups. The lipid molecules form a
bilayer around the DNA molecules, which creates liposomes that are mixed with cells. Cells
take up the lipid-DNA molecules and some of the DNA enters the nucleus.
- Viral vectors: the introduction of new genes into mammalian cells by packaging them into
virions is called viral gene transduction. The virions used for this purpose are called viral
vectors.
o Adenovirus: Adenoviruses are non-enveloped, linear, double-stranded DNA viruses.
Adenoviral vectors have potential advantages over retroviral vectors. Adenoviruses
can deliver transgenes to both dividing and nondividing cells, and adenoviruses are
relatively stable and resistant to physical manipulations and can be frozen for use at
later times. An disadvantage is the induction of a significant host immune response
and the development of virus-neutralizing antibodies. It has a low cloning capacity.
Their random integration in the genome might disturb the normal expression of
cellular genes encoding proteins regulating cellular replication.
o Adeno-associated virus (AAV): small, single-stranded, non-pathogenic DNA viruses.
AAVs require a helper virus for replication and completion of life cycle. AAVs are
composed of three genes: rep, which is responsible for viral DNA replication, and
cap, which packages the viral genome. Therapeutic expression cassette replaces rep
and cap, leaving the viral inverted terminal repeats (ITRs) as the only viral sequences.
o Retrovirus: LTR retroviruses are single-stranded RNA viruses that consist of the 5’
and 3’ long-terminal repeats and the gag, pol and env genes. Upon interaction with
specific host-cell membrane proteins the retroviral envelope fuses with the plasma
membrane. 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. Because most retroviruses do not kill their host-cells, infected cells
can replicate, producing daughter cells with integrated proviral DNA. The packaging
sequence is needed to confer packaging when all of the structural genes are
expressed by the helper cell.
Packaging cell line: provide all the viral proteins required for capsid
production and virion maturation of the vector. These packaging cell lines
have been made so that they contain the gag, pol and env genes.
Micro-injection in fertilized egg: fertilised eggs are collected by washing out the oviducts of mated
females, and the gene of interest is injected into one of the two pronuclei. The injected eggs are
transferred to foster mothers, female mice made pseudopregnant by mating with vasectomized
males. The offspring are checked for the presence of the transgene by Southern blotting of DNA
extracted from a small piece of the tail. It is mainly used when aiming at the production of a
transgenic mouse that over-expresses a gene of interest. The transgenic construct at a minimum
contains a promoter element, complementary DNA for the gene of interest and a polyadenylation
signal.
Totipotent: when a sperm cell and an egg cell unite, they form a one-celled fertilized egg. This cell is
totipotent, meaning that it has the potential to give rise to any and all human cells. It can even give
rise to an entire functional organism.
Pluripotent: embryonic stem (ES) cells from the inner cells of the blastocyst are like totipotent stem
cells in that they can give rise to all tissue types. Unlike totipotent stem cells, however, they cannot
give rise to an entire organism.
Multipotent: these more differentiated stem cells give rise to a limited range of cells within a tissue
type or organ. An adult (or somatic) stem cell is a multipotent stem cell in adult humans that is used
to replace cells that have died or lost function. It is an undifferentiated cell present in differentiated
tissue.
Transgenic: mice that carry the foreign gene.
Transgene: foreign DNA.
Gene therapy: an experimental technique that uses genes to treat or prevent diseases.
- Direct delivery: The therapeutic gene is packaged into a delivery vehicle such as a retrovirus
and injected into the patient (for example in a target organ).
- Stem cell-based delivery: The therapeutic gene is packaged into a delivery vehicle such as a
retrovirus and introduced into the cells (from the patient, ex vivo admission). The genetically
modified cells are reintroduced into the patient (for example in a target organ).
Stem cells: self-renewing cells that have the potential to generate different cell types.
- Embryonic stem cells: DNA can be introduced into ES cells by electroporation, lipofection or
transfection. Their most important advantage for gene transfer into mice is that ES cells
carrying the transgene can be selected for, before being injected into a blastocyst.
- Induced pluripotent stem cells: iPSC are derived from skin or blood cells that have been
reprogrammed back into an embryonic-like pluripotent state that enables the development
of an unlimited source of any type of human cell needed for therapeutic purposes.
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