Gene Knockouts, Transgenics, and Genome Editing

Question 1

an organism that gains new genetic information from the addition of foreign DNA is called _______

Answer 1

transgenic

Question 2

gene deletions are usually referred to as ____________, whereas replacement of a gene with an alternative mutated version is called a _____________

Answer 2

knockouts
knock-in

Question 3

reduce the amount of a gene product (RNA or protein) produced via RNA interference to selectively target specific mRNAs for degradation

Answer 3

knockdown

Question 4

the introduction of a novel gene from one organism into the genome of another that potentially change the phenotype of an organism

Answer 4

transgenesis

Question 5

an experimentally introduced DNA segment carried in the genome of a host animal

Answer 5

transgene

Question 6

what can a transgene be designed to do?

Answer 6

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

Question 7

Transgenesis will change the germ cells to ensure what?

Answer 7

the transgenes are passed down to the offspring when the organisms reproduce

Question 8

what type of injection is the transgene delivered into the mouse embryo

Answer 8

pronuclear injection

Question 9

_______________ are modified and the targeted _______ are injected into mouse blastocysts

Answer 9

embryonic stem cells

Question 10

describe how transfection can introduce DNA directly into the germline of animals.

Answer 10

-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

Question 11

describe how ES cells can be used to generate mouse chimeras:

Answer 11

-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

Question 12

knocking out a gene means to mutate the DNA in a way that ____________________

Answer 12

ceases the gene expression permanently

Question 13

a site-specific recombinase technique to carry out inducible knockout at specific sites

Answer 13

Cre/lox recombination

Question 14

catalyzes efficient excision recombination in mammalian cells and has been become a useful tool for generating a conditional KO

Answer 14

Cre recombinase of phage P1

Question 15

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

Answer 15

Cre recombinase

Question 16

what is the great utility of the Cre/lox system

Answer 16

it requires only the Cre enzyme produced in any cell that has a pair of lox sites

Question 17

how can cre/lox be regulated to work in a particular cell

Answer 17

placing the cre gene under control of a regulated promoter

Question 18

describe the cre/lox process

Answer 18

-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

Question 19

can be activated in an adult organism, where the activity of the gene can be turned on or off

Answer 19

conditional gene knockout

Question 20

the offspring of the cre/lox cross in which the function of the gene is lost only in cells that express Cre

Answer 20

conditional knockouts

Question 21

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

Answer 21

genome-editing

Question 22

-nucleic acid cleaving enzymes that can be programmed and used for genetic manipulation
-4 classes

Answer 22

nucleases

Question 23

nucleases derived from bacteria

Answer 23

meganucleases

Question 24

nucleases based on eukaryotic transcription factors

Answer 24

Zinc finger nucleases

Question 25

What are TALENs

Answer 25

transcription activator-like effector nucleases from bacteria

Question 26

how do nucleases recognize their DNA binding site

Answer 26

protein-DNA interaction

Question 27

Genome editing techniques based on meganucleases, ZFN, or TALENs uses _______________________________ to introduce a piece of exogenous DNA

Answer 27

homologous recombination and double strand breaks

Question 28

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

Answer 28

ZFNs

Question 29

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

Answer 29

TALENs

Question 30

targeted to DNA sequences complementary to the targeting sequence within the single guide RNA (gRNA)

Answer 30

Cas9 nuclease

Question 31

How do the DSBs introduced by endonucleases effect genome editing outcomes?

Answer 31

they drive activation of cellular DNA repair pathways and facilitate the introduction of site-specific genome modfications

Question 32

gene knockout via random base insertions and/or deletions can be introduced by__________________

Answer 32

nonhomologous end joining (NHEJ)

Question 33

what can occur in the presence of a donor template with homology to the targeted chromosomal site, gene integration, or base correction

Answer 33

homology-directed repair (HDR)

Question 34

targeted nucleases induce ________________ that are repaired by _____________or in the presence of donor template, ________________

Answer 34

DSBs
NHEJ
HDR

Question 35

what does NHEJ introduce in absence of a donor template?

Answer 35

small base insertions or deletions that can result in gene disruptions

Question 36

When 2 DSBs are induced simultaneously, the intervening genomic sequence can be _________________

Answer 36

deleted or inverted

Question 37

in the presence of donor DNA, recombination between homologous DNA sequences present on the donor template and a specific chromosomal site can facilitate _________________

Answer 37

targeted integration