woordenlijst Flashcards
Starting and maintaining a cell line
starting a cell line:
- isolate individual cells by disrupting the extracellular matrix and cell-junctions
- mammalian cells require a solid surface that is coated with material they can adhere to
cell cultures directly preparen from tissue of an organism are primary cell cultures. repeated culturing can result in a cell line (immortalized my mutations). cell lines can be most easily generated from cancer cells. growth factors are needed to stimulate replication of specific cell types, and they can maintain a specific differentiated cell status
recombination based (gateway)
gateway cloning is based on the highly specific integration and excision reactions of bacteriophage λ into and out of the E. coli genome. the gateway cloning system uses two different enzyme mixtures each of which performs a different type of recombination reaction. the BP clonase enzyme mix recombines attB sites with attP sites, generating attL and attR sites; whereas the LR clonase enzyme mix catalyzes the reverse reaction. each recombination event is precise, with no nucleotides either gained or lost.
dna sequences to be cloned are added to modified versions of these special Gateway Att sites. two enzyme reactions take place, BP clonase and LR clonase. the BP clonase occurs between the attB sites surrounding the insert and the attP sites of the donor vector. this reaction is catalyzed by the BP clonase enzyme mixture and produces the entry clone containing the DNA of interest flanked by attL domains. as a byproduct of the reaction, the ccdB gene is cut from the donor vector. the LR clonase occurs between the attL regions of the generated entry clone and the attR regions of the destination vector and is catalyzed by the LR clonase enzyme mix. as a result, an expression clone with dna of interest flanked by attB regions is produced (expression clone). as in the BP reaction, a DNA sequence containing the ccdB gene is cut from the target vector
golden gate cloning (type-IIs restriction enzymes
golden gate cloning is a moleculat cloning method that allows a researcher to simultaneously and directionally assemble multiple DNA fragments into a single piece using Type IIS restriction enzymes (Bsal) and T4 DNA ligase. these enzymes cut DNA outside of their recognition sites and, therefore, can create non-palindromic overhangs (on + or - strand). golden gate cloning is tapically scarless. additionally, because the final product does not have a type IIS restriction enzyme recognition site, the correctly-ligated product cannot be cut again by the restriction enzyme, meaning the reaction is essentially irreversible. this means that it is possible to do the digestion and ligation of the DNA fragments in a single reaction, whereas in conventional cloning methods these reactions are separate
modular cloning (introducing multiple genes)
co-transformation of multiple plasmids. restriction ligation cloning of 2 or more genes in a singly binary vector. limited by complex restriction-ligation reactions. alternative: recombinase-based cloning (multisite gateway), type IIS restriction enzymes (golden gate cloning) or synthetic biology
synthetic biology
design and construct of new biological parts devices and system and re-design of existing, natural biological systems for useful purposes. it encompasses a variety of different approaches, methodologies, and disciplines with a focus on engineering biology and biotech. synthetic biologists approach the creation of new biological systems from different perspectives, focusing on finding how life works or how to use it to benefit society
engineering bio-synthetic pathway in plants (golden rice)
increased levels of pro-vitamin A (beta-carotene). beta-carotene is essential precursor for human vitamin A production. normal rice does not contain beta-carotene in endosperm. millions suffer from vitamin A deficiencies. transfer 3 genes to modulate the beta-carotene biosynthetic pathway
insect targeted RNAi in plant chloroplasts (inducible RNAi in plants)
feeding insects dsRNAs that target essential genes through the RNAi mechanism can result in death of the insect. chloroplasts lack the cellular RNAi machinery and therefore, lon dsRNA are not cleaved by Dicer into siRNA
glyphosate-resistant weeds
overuse of BT and HT crops result in evolution of resistance. the number glyphosate-resistant weeds is accelerating, due to: Horizontal (trans)gene transfer; target site mechanism; non-target site mechanism; mechanisms that restrict glyphosate movement
herbicide (glyphosate) tolerant crop
glyphosate herbicide kills plants by blocking the EPSPS enzyme, an enzyme involved in the biosynthesis of aromatic amino acids, vitamins and many secondary plant metabolites. there are several ways by which crops can be modified to be glyphosate-tolerant. one strategy is to incorporate a soil bacteriom gene that produces a glyphosate tolerant form of EPSPS. another way is to incorporate a different soil bacterium gene that produces a glyphosate degrading enzyme (strategy abandoned). glyphosate is an active ingedrient in the commercial weed killer Roundup. it is active in low doses against wide spectrum of weeds
insect resistant crop (BT-crop)
bacillus thuringiensis (Bt) crops are plants genetically engineered (modified) to contain the endospore (or crystal) toxins of the bacterium, Bt to be resistant to certain insect pests. insect resistance converted by Bt cry proteins natural microbial pesticides, such as Bacillus thuringiensis, have been used in limited fashoin. upon sporulation, bacteria produce a crystallized protein (Cry protein) that is toxic to insects. toxin protein (Bt-toxin) normally expressed as a large, inactive pro-toxin of about 1200 amino acids. upon digestion by susceptible larvae, proteases in the insect gut cleave the pro-toxin into an active 600 amino acids fragment. activated toxin binds to receptors on surface of midgut cells, making it permeable to ions and small molecules so that the cell bursts. Bt is effective only when eaten by the insect as larva. since Bt is applied topically insects that attack the roots or the insides of a plant will not be affected. Bt-toxin genes introduced in plants. Bt-toxin genes are AT-rich compared plant genes, causing: premature transcription termination because of AAUAAA; aberrant mRNA splicing; Cryptic mRNA splice sites; mRNA instability; inefficient codon usage. therefore, partially synthetic (codon optimized) Bt-toxin genes constructed
gene drive
a gene drive is a self-propagating mechanism by which a desired genetic variant can be spread through a population faster than traditional Mendelian inheritance. this strategy can be so effective that alleles can spread even if they confer a disadvantageous trait
CRISPR-Cas9
CRISPR-Cas9 edits genes by precisely cutting target DNA complementary to the complementary RNA guide and then letting natural DNA repair processes to take over. the system consists of two parts: the Cas9 enzyme and a guide RNA. it cuts before the PAM site after RNA-DNA hybrid is formed after nuclease is lead to the site by the RNA strand
TALE Nucleases
TALENs are chimeric proteins that contain two functional domains: a DNA-recognition transcription activator-like affedtor (TALE) and a nuclease domain. they work for gene editing by recognizing a specific sequence, which the user can design, and introducing a double-stranded break with an overhang, TALENs therefore serve as a form of customizable restriction enzyme. also fused to the cleavage domain of Fokl. one binding subunit binds 1 nucleotide
zinc finger nucleases
a ZFN is an artificial endonuclease that consists of a designed zinc finger protein (ZFP) fused to the cleavage domain of the Fokl restriction enzyme. a ZFN may be redesigned to cleave new targets by developing ZFPs with new sequence specifities. one binding subunit binds 3 nucleatide for genome engineering, a ZFN is targeted to cleave a chosen genomic sequence. the cleavage event induced bu the ZFN provokes cellular repair processes that in turn mediate efficient modification of the targeted locus. it can either result in deletions or insertions
Cre-LoxP recombination system (and FRT-FLP system)
used for site spedific recombination events. first homologous recombination to create LoxP sites with neomycin in system. Cre recombinase (from bacteria P1) is responsible for recombination at the LoxP recognition sites. Cre recognizes two directly repeated LoxP sites. Cre then excises the LoxP flanked DNA, creating two types of DNA: circular excised and inactivated Y gene. one LoxP site stays in inactivated gene upon knockout.
the FLP-FRT system is similar to the Cre-lox system and is becoming more frequently used in mouse-based research. it involves using flippase (FLP) recombinase, derived from the yeast Saccharomyces cerevisiae. FLP recognizes a pair of FLP recombinase target (FRT) sequences that flank a genomic region of interest
Fluorescence-activated cell sorter
Antibody coupled to a fluorescent dye to label specific cells.
Droplets containing single cells are given an negative or
positive charge, depending on whether the cell is
fluorescent. And deflected by an electrical field into
collection tubes according to their charge.
Ca2+ phosphate coprecipitation
DNA and calcium phosphate are allowed to form a
precipitate that is then added to cells in culture. The cells
internalize the DNA, through endocytosis, leading to the
expression of the transfected genes in the cell.
Electroporation
An electrical pulse is used to create temporary pores in cell
membranes through which substances can pass. After
electroporation, the cell membrane recovers, and expression
of the transfected nucleic acid can occur.
Lipofection
The use of a lipid complex, liposomes, to deliver DNA to
cells. The liposomes have the same composition as the cell
membrane.
Viral vectors
Infection by membrane fusion, pore formation or membrane
disruption. Maintained in the cell nucleus by integration in
the host genome or as episome
Adenovirus
Adenoviruses most commonly cause respiratory illness.
Adenovirus (Ad) is a nonenveloped, dsDNA virus. Viral DNA
and associated core proteins are encased in an icosahedral
capsid. The virus enters the cell in a clathrin-coated vesicle
and is transported to endosomes, where acidification results
in partial disassembly of the capsid; the altered virion
escapes into the cytoplasm and is transported to the
nucleus, where replication occurs
Adeno-associated
virus (AAV)
ssDNA
Gene therapy vectors using AAV can infect both dividing and
quiescent cells and persist in an extrachromosomal state
without integrating into the genome of the host cell.
Replication defective; Lack of pathogenicity; Does not
generally integrate in the genome. To generate recombinant
AAV, a gene is inserted between the ITRs to replace both rep and cap. Rep is essential for replication, transcriptional
regulation and virus particle assembly. Cap for viral capsid
proteins.
Retrovirus (inc.
lifecycle)
A retrovirus is a virus that uses RNA as its genomic material.
Upon infection with a retrovirus, a cell converts the
retroviral RNA into DNA (using reverse transcriptase), which
in turn is inserted into the DNA of the host cell. Needs cis
elements for packaging, reverse transcription and
integration. The cell then produces more retroviruses, which
infect other cells. Disadvantages: Low cloning capacity;
Eukaryotic cells should divide to enable integration; Cannot
control integration site in host genome. Self-inactivating
(SIN) retroviral vector: Mutated 3’LTR results in promoterless
insert upon viral DNA integration; Expressed as plasmid;
Expression after regulation.
Micro-injection in fertilized egg
(mouse)
Used to add new (extra) genetic information (cloned gene)
to existing gene. DNA microinjection is the dominating
technique leading to random integration of a transgene via
the introduction of DNA into the pronucleus of a developing
zygote. Following fertilization of a mouse egg, the male and
female pronuclei remain separated for a few hours before
they fuse to make the zygotic nucleus, thus allowing for
microinjection of the desired genes into the larger male
pronucleus. Eggs that can survive the injections are
transferred into the oviducts of a foster mother i.e.,
pseudopregnant female mice for the generation of Founder
mouse, from which permanent transgenic lines can be
established. The presence of transgenes in the offsprings can
be identified by PCR analysis or Southern blot hybridization.
+ Highly efficient with mice (10-30%)
+ Transgenic progeny is heterozygous (and not chimeric) for
transgene.
+ No selection marker needed.
Random integration of DNA in genome.
Low efficiencies in other species than rodent
Gene therapy
Gene therapy is a medical approach that treats or prevents
disease by correcting the underlying genetic problem. Gene
therapy techniques allow doctors to treat a disorder by
altering a person’s genetic makeup by gene addition, gene
correction/alteration or gene knockdown.
Direct delivery
In direct delivery the therapeutic gene is packaged into a
delivery vehicle, such as a retrovirus, and injected into the
patient (e.g. liver). It can also be delivered by lipofection.
