Resume
Summary Gene Technology: all Lectures & Cases
- Cours
- Établissement
- Book
Summary of the course Gene technology, including an overview with all lectures, topics and the online cases. Also includes illustrations and schemes
[Montrer plus]
Livre connecté
Titre de l’ouvrage:
Auteur(s):
- Édition:
- ISBN:
- Édition:
Plus de résumés pour
-
Molecular Biology of The Cell, 7th Edition
-
Samenvatting van de hoofdstukken 4, 5, 6 en deels 7 (t/m 7.3) - Cellen en weefsels
-
Summary Molecular Biology of the Cell 2 (book) (WBFA007-04)
Vendeur
S'abonner
merelhendrikx
Avis reçus
Aperçu du contenu
Lecture 1: Gene transfer to mammaliancells
Gene transfer to Mammalian cells
1. Isolate & clone gene of interest (cloned in plasmids and multiplied/manipulated ALWAYS in
E. coli)
2. Manipulate sequence of gene in vitro
3. Return the altered gene back into organism or single cells
Gene transfer has become a routine tool for studying gene structure and function: it is used to
identify the regulatory sequences that control gene expression
Why gene transfer to mammalian cells?
• Research – Study gene function: through knockout, over-expression or reporter gene
(localization), provide evidence
• Biotechnology – use cells as bioreactor: transgenic animals will produce protein of interest
• Gene therapy: insertion of a gene into an individual’s cells and tissues to treat a hereditable
disease whereby a deleterious mutant allele is replaced with a functional gene
Return altered gene into Single cells
Starting a cell line
Primary cell cultures: Cell cultures directly prepared from tissues of an organism
Secondary cell cultures: cell cultures that are sub-cultured from a primary culture
Cell strain: a lineage of cells originating from one initial primary culture with a limited lifetime
Cell line: immortal cells as they underwent some genetic changes allowing them to grow unlimited
Starting and maintaining a cell line:
• Isolate individual cells by disrupting the extracellular matrix and cell-junctions: dissociate
cells from tissues
o Proteolytic enzymes trypsin & collagenase to digest/destroy matrix
o EDTA solution to chelate Ca2+ on which cell-cell adhesion depends
• Mammalian cells require a solid surface that is coated with material they can adhere to in
the Petri dish
o e.g., polylysine or extracellular matrix components
o This property can be used to obtain specific cell types based on surface properties
and their binding to antibodies (e.g., FACS – antibodies for specific cells)
, ▪ Some cells bind specifically to specific coatings → sort → isolate specific
cells for a specific cell line
• Cell lines can most easily be generated from tumors/cancer cells.
o They are capable of indefinite replication (unlimited cell divisions) in culture and
express at least some of the special characteristics of their cells of origin.
• Growth factors are needed to stimulate replication of specific cell types
o Cells in culture remain differentiated
• Repeated cell culturing can result in a cell line → immortalized by mutations
Techniques to sort cells
FACS: Fluorescence-activated cell sorter → Cells are sorted based on their fluorescence
• Antibody bound to cells & labeled with fluorescent dye to label specific cells/epitopes
• Droplets containing single cells are given a negative or positive charge, depending on
whether the cell is fluorescent
• Delivers purified cells
o And deflected by an electric field into collection tubes according to their charge
• Start a very specific cell culture
Micro-dissection from tissue slices
• Cells are recognized morphologically (shape/size) that you want to isolate
• Laser beam cuts around region of interest cells and can be dropped into an Eppendorf to be
cultured
• Gravity or second laser to obtain cells
Methods for introducing DNA constructs into mammalian cells: transformation
methods
1. Ca2+ phosphate co-precipitation: Get DNA into cells (mammalian/bacteria)
a. Cells efficiently take up DNA when it is in the form of a precipitate with calcium
phosphate → divalent cations (Ca2+/Mg2+) promote DNA uptake into bacteria
b. DNA contains lot of phosphate → calcium + DNA → DNA precipitate → add DNA
precipitate to cells → DNA is taken up by the cell
2. Electroporation: Apply voltage
a. Mix cells and DNA → Electric shocks on cells → holes in membrane → Cells take up
DNA into cytoplasm directly through holes in membrane (made through shocks) →
integration in genome of cell
i. Not through vesicles, as passage through the endocytic vesicles might
destroy or damage DNA
, b. Stable expression means integration in the genome so that the DNA persists in all
cells derived from the initial few cells that were transformed (linear DNA) – selection
i. Transient expression: Circular DNA less likely to integrate in the DNA
c. No salts: will kill de cell – apply rich medium: take care that the cells will survive –
apply selection medium: stable expression (linear DNA)
3. Lipofection: DNA is packed in a micelle/liposome. Plasmid-liposome complex-mediated gene
transfer
a. DNA (-) is mixed with a lipid solution (+) → micelles are formed → attach to cells (-)
and fuse into plasma membrane (endocytosis) → DNA inserted in
cytoplasm → integrated in the genome → selection
b. Advantages:
i. Lack proteins → non-immunogenic
ii. Can carry exogenous material of essentially unlimited size
iii. Cannot replicate/recombine to form infection agents
c. Disadvantages:
i. Low transduction efficiency as compared to viral vectors
4. Viral vectors: Infect cells efficiently by membrane fusion, pore formation or
membrane disruption – non-lytic
a. Adenovirus (ds DNA virus)
b. Adeno-associated virus (ss DNA virus)
c. Retrovirus (ss RNA virus)
Figure 1. Gene transfer by Ca2+ phosphate co-precipitation
, Viral vectors
Viral gene transduction: introduction of new genes into mammalian cells by packaging them into
virions (= viral vector) The virus is used to insert the transgene into a cell instead of their own genes.
• Infection: Infect cells efficiently by
o Membrane fusion – pore formation –
(endosomal) membrane disruption
o Natural route of entry
• Transfection: introducing nucleic acids into eukaryotic
cells by nonviral methods
• Non-lytic enveloped viruses
o Non-enveloped viruses usually leave an infected
cell by lysing it, but non-lytic enveloped viruses
do not kill the cell → Cell remains alive
o Viruses with an envelope do not kill the cell
when they leave → keep the cell alive and keep
producing
• Maintained in cell nucleus: integrate in the host genome
or as episome
o Long terminal repeats (LTR) needed for
integration
From virus to viral vector:
- The number of nucleic acids that can be inserted in the virus particle
is fixed
o You cannot make the genome size of the virus larger
o When you want to insert your transgene into a virus
particle, other viral genes have to be taken out
- Those viruses should not trigger the immune system of the
host/patient
o Viruses that can escape from the immune system are
preferred
o Coat proteins of virus trigger the immune system
- Some viruses integrate in the genome whereas other stay out of the
genome (episomes)
Viral vectors:
• DNA Adenovirus: maintained as extrachromosomal dsDNA, non-enveloped
o DNA in nucleus as extracellular molecule (episomes)
o Advantages over retroviral vectors:
▪ Most human cell types are susceptible to adenoviral infection and are
subject to efficient transduction
▪ Infects and replicates in dividing and non-dividing cells
▪ Stable and resistant to physical manipulations
▪ Adenoviral cycle does not require integration into the host genome
▪ Promotor of choice can be used (tissue selective expression)
Les avantages d'acheter des résumés chez Stuvia:
Qualité garantie par les avis des clients
Les clients de Stuvia ont évalués plus de 700 000 résumés. C'est comme ça que vous savez que vous achetez les meilleurs documents.
L’achat facile et rapide
Vous pouvez payer rapidement avec iDeal, carte de crédit ou Stuvia-crédit pour les résumés. Il n'y a pas d'adhésion nécessaire.
Focus sur l’essentiel
Vos camarades écrivent eux-mêmes les notes d’étude, c’est pourquoi les documents sont toujours fiables et à jour. Cela garantit que vous arrivez rapidement au coeur du matériel.
Foire aux questions
Qu'est-ce que j'obtiens en achetant ce document ?
Vous obtenez un PDF, disponible immédiatement après votre achat. Le document acheté est accessible à tout moment, n'importe où et indéfiniment via votre profil.
Garantie de remboursement : comment ça marche ?
Notre garantie de satisfaction garantit que vous trouverez toujours un document d'étude qui vous convient. Vous remplissez un formulaire et notre équipe du service client s'occupe du reste.
Auprès de qui est-ce que j'achète ce résumé ?
Stuvia est une place de marché. Alors, vous n'achetez donc pas ce document chez nous, mais auprès du vendeur merelhendrikx. Stuvia facilite les paiements au vendeur.
Est-ce que j'aurai un abonnement?
Non, vous n'achetez ce résumé que pour €5,79. Vous n'êtes lié à rien après votre achat.
Peut-on faire confiance à Stuvia ?
4.6 étoiles sur Google & Trustpilot (+1000 avis)
80467 résumés ont été vendus ces 30 derniers jours
Fondée en 2010, la référence pour acheter des résumés depuis déjà 14 ans