Topic Lecture
Starting and maintaining a Cell line 1
- Isolate cells by disrupting extracellular matrix and cell
junctions o Trypsin and collegonase o EDTA solution to
chelate Ca2+
- Require solid surface to adhere to (polylysine or extracellular
components o Used to obtain specific cell types
- Mostly generated from cancer cells
- Primary cell cultures are prepared directly from tissues
Fluorescence-activated cell sorter 1
- Antibody coupled fluorescence to label specific cells
- The sorter sorts the cells by charging them positively or
negatively, after which they are deflected and collected
Laser micro-dissection microscope 1
- Tool for selecting specific area with cells by laser cutting
followed by ‘laser-catapult’
Transformation methods for animal cells 1
* Ca2+ phosphate co-precipitation 1
- DNA (from adeno-virus) dissolved in phosphate buffer in
which DNA precipitates after CaCl2 is added. The DNA is
introduced to cells, which start producing the virus with the
wanted DNA.
* electroporation 1
- DNA and cells are mixed and treated with heat-shock, the
DNA is absorbed by the cells and nucleus and can be
selected on expression gene
* lipofection 1
- DNA is packed in lipid bilayer (liposomes) and introduced to
cells. Liposomes fuse with membrane cell and DNA goes
into the nucleus
viral vectors 1
- adenovirus
- dsDNA virus in linear episomes
- not used too much -> vector toxicity 1
- adeno-associated virus (AAV) 1
- ssDNA virus in circular and linear episomes
- small capacity of DNA (4.7 kb)
- retrovirus (inc. lifecycle of retrovirus) 1
- ssRNA with high selectivity
- integrates DNA into genome of host, after RNA is reverse
transcribed into DNA -> cell keeps producing virus (like HIV)
- encodes for gag (capsid proteins), env (envelope protein)
and pol (reverse transciptase)
, lOMoAR cPSD| 22218783
- packaging cell line
- Cell line used to produce (package) viral vectors with an
inserted gene
- This is done by inserting gene of interest bordered by LTR’s,
which are needed for integration into virus 1
Micro-injection in fertilized egg (mouse) 1
- Gene of interest is inserted into pronucleus, which is
transferred to a pseudopregnant mouse, after the offspring is
selected by DNA analysis to confirm gene uptake
- High efficiency, only in rodents, not humans
Gene therapy 1
- In vivo/ex vivo
* direct delivery 1
- In vivo -> viral or non viral vectors (AAV, Adeno-virus,
lipofection)
* stem cell-based delivery 1
- Gene introduced in stemcells, then put back in to target
tissue
stem cells 1
- Self renewing, potential to generate different cell types
- Totipotent: can form any cell, even entire organism
- Pluripotent: can form any cell
- Multipotent: limited range of formed cell types
* embryonic stem cells (mouse) 1
- Can be maintained indefinitely and used to produce different
cells (pluripotent cells)
* induced pluripotent stem cells 1
- Pluripotent cells made from somatic cells, by forcing the
expression of 4 genes (OSKM)
Reporter genes 2
- Genes that inserted to report whether the gene has been
integrated and is activated
- Transcriptional reporter: to determine promotor activity
- Translational reporter: to determine protein localization (&
activity)
o Can be used to discover cis-regelatory elements
* luciferase
- Codes for an enzyme that produces a product from a
fluorescent substrate (luciferin, from fireflies) that can be
detected.
- GUS produces plue precipitate from carbohydrates
- LacZ -> blue reporter after substrate (x-gl)
- INDIRECT DETECTION 2
* GFP / DsRED / eCFP/ eYFP
- Fluorescent proteins that can be inserted
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