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
Voordelen van het kopen van samenvattingen bij Stuvia op een rij:
Verzekerd van kwaliteit door reviews
Stuvia-klanten hebben meer dan 700.000 samenvattingen beoordeeld. Zo weet je zeker dat je de beste documenten koopt!
Snel en makkelijk kopen
Je betaalt supersnel en eenmalig met iDeal, creditcard of Stuvia-tegoed voor de samenvatting. Zonder lidmaatschap.
Focus op de essentie
Samenvattingen worden geschreven voor en door anderen. Daarom zijn de samenvattingen altijd betrouwbaar en actueel. Zo kom je snel tot de kern!
Veelgestelde vragen
Wat krijg ik als ik dit document koop?
Je krijgt een PDF, die direct beschikbaar is na je aankoop. Het gekochte document is altijd, overal en oneindig toegankelijk via je profiel.
Tevredenheidsgarantie: hoe werkt dat?
Onze tevredenheidsgarantie zorgt ervoor dat je altijd een studiedocument vindt dat goed bij je past. Je vult een formulier in en onze klantenservice regelt de rest.
Van wie koop ik deze samenvatting?
Stuvia is een marktplaats, je koop dit document dus niet van ons, maar van verkoper hamzalaasri. Stuvia faciliteert de betaling aan de verkoper.
Zit ik meteen vast aan een abonnement?
Nee, je koopt alleen deze samenvatting voor €7,85. Je zit daarna nergens aan vast.
