Gene Knockouts, Transgenics, and Genome Editing Flashcards
an organism that gains new genetic information from the addition of foreign DNA is called _______
transgenic
gene deletions are usually referred to as ____________, whereas replacement of a gene with an alternative mutated version is called a _____________
knockouts
knock-in
reduce the amount of a gene product (RNA or protein) produced via RNA interference to selectively target specific mRNAs for degradation
knockdown
the introduction of a novel gene from one organism into the genome of another that potentially change the phenotype of an organism
transgenesis
an experimentally introduced DNA segment carried in the genome of a host animal
transgene
what can a transgene be designed to do?
encode a new gene product in the transgenic animal, or it can be introduced with the intent of altering or disrupting a host gene at its site of insertion
Transgenesis will change the germ cells to ensure what?
the transgenes are passed down to the offspring when the organisms reproduce
what type of injection is the transgene delivered into the mouse embryo
pronuclear injection
_______________ are modified and the targeted _______ are injected into mouse blastocysts
embryonic stem cells
describe how transfection can introduce DNA directly into the germline of animals.
-plasmids injected into the nucleus of oocyte or pronucleus of fertilized egg
-egg implanted into pseudopregnant mouse
-after birth, recipient mouse examined so see if it has expressed the foreign DNA
describe how ES cells can be used to generate mouse chimeras:
-ES cells derived from mouse blastocyst
-genes added to mouse germline by transfecting them into ES cells before they are added to blastocyst
-ES cells that are injected into blastocyst generate descendant cells that become part of a chimeric adult mouse
knocking out a gene means to mutate the DNA in a way that ____________________
ceases the gene expression permanently
a site-specific recombinase technique to carry out inducible knockout at specific sites
Cre/lox recombination
catalyzes efficient excision recombination in mammalian cells and has been become a useful tool for generating a conditional KO
Cre recombinase of phage P1
an enzyme that recognizes the specific DNA fragment sequences called lox P site and mediates site-specific deletion of DNA sequences between two lox P sites
Cre recombinase
what is the great utility of the Cre/lox system
it requires only the Cre enzyme produced in any cell that has a pair of lox sites
how can cre/lox be regulated to work in a particular cell
placing the cre gene under control of a regulated promoter
describe the cre/lox process
-cre recombinase recognizes loxP sites of specific DNS sequences
-gene of interest is flanked in two loxP sites
-cre recombinase catalyzes a site-specific recombination between two identical lox sites then cre excises loxP flanked DNA, releasing the DNA and inactivating the gene
can be activated in an adult organism, where the activity of the gene can be turned on or off
conditional gene knockout
the offspring of the cre/lox cross in which the function of the gene is lost only in cells that express Cre
conditional knockouts
The highly versatile ____________________technologies has provided the ability to rapidly and economically introduce sequence-specific modifications into the genomes of a broad spectrum of cell types and organisms
genome-editing
-nucleic acid cleaving enzymes that can be programmed and used for genetic manipulation
-4 classes
nucleases
nucleases derived from bacteria
meganucleases
nucleases based on eukaryotic transcription factors
Zinc finger nucleases
What are TALENs
transcription activator-like effector nucleases from bacteria
how do nucleases recognize their DNA binding site
protein-DNA interaction
Genome editing techniques based on meganucleases, ZFN, or TALENs uses _______________________________ to introduce a piece of exogenous DNA
homologous recombination and double strand breaks
recognize target sites that consist of two zinc-finger binding sites that flank a 5- to 7-base pair spacer sequence recognized by the Fokl cleavage domain
ZFNs
recognize target sites that consist of two TALE DNA-binding sites that flank a 12- to 20- bp spacer sequence recognized by the Fokl cleavage domain
TALENs
targeted to DNA sequences complementary to the targeting sequence within the single guide RNA (gRNA)
Cas9 nuclease
How do the DSBs introduced by endonucleases effect genome editing outcomes?
they drive activation of cellular DNA repair pathways and facilitate the introduction of site-specific genome modfications
gene knockout via random base insertions and/or deletions can be introduced by__________________
nonhomologous end joining (NHEJ)
what can occur in the presence of a donor template with homology to the targeted chromosomal site, gene integration, or base correction
homology-directed repair (HDR)
targeted nucleases induce ________________ that are repaired by _____________or in the presence of donor template, ________________
DSBs
NHEJ
HDR
what does NHEJ introduce in absence of a donor template?
small base insertions or deletions that can result in gene disruptions
When 2 DSBs are induced simultaneously, the intervening genomic sequence can be _________________
deleted or inverted
in the presence of donor DNA, recombination between homologous DNA sequences present on the donor template and a specific chromosomal site can facilitate _________________
targeted integration
