1- transgenesis

Question 1

what are transgenic or genetically modified animal lines

Answer 1

genome has been stably altered in all cells, including cells that form germline

Question 2

germ cells are _____ cells

Answer 2

haploid

Question 3

somatic cells

Answer 3

non-germ cells that do not contribute to subsequent generations

Question 4

Embryonic stem cells

Answer 4

derived from undifferentiated cells of blastocyst ICM and can be pluripotent. they also can be maintained in culture and retain the ability to form all differentiated cells

Question 5

induced pluripotent stem cells

Answer 5

somatic cells that is made into ES cell via reprogramming with ES cell genes

Question 6

chimera

Answer 6

an animal containing cells from 2 different individuals

Question 7

tissue specific stem cells regenerate what

Answer 7

specific tissues

Question 8

what is transgenesis

Answer 8

1. modification of germ cells
2. permanent alteration of genome
3. offspring can inherit

Question 9

what is gene therapy

Answer 9

1. modification of somatic cells
2. alteration can be stable or transient
3. does nothing for future generations

Question 10

steps to embryo dev

Answer 10

1. fertilized egg
2. morula
3. blastocyst
4. early blastocyst with ICM
5. embryo

Question 11

what methods to produce transgenic animals

Answer 11

1. microinjection or DNA into fertilized eggs/pronuclear injection of DNA
2. gene targeting in ES cells
3. nuclear transfer from genetically altered cells (cloning)
4. DNA editing using site directed nucleases

Question 12

does microinjection of DNA use germ or somatic cells

Answer 12

germ cell

Question 13

does gene targeting use germ or stem cell

Answer 13

stem cell

Question 14

does nuclear transfer from genetically altered cells use somatic or germ cells

Answer 14

can use both

Question 15

does dna editing use germ or somatic cell

Answer 15

germ

Question 16

what do you inject in the first method (pronuclear injection)

Answer 16

promoter
cDNA (coding) : AKA only exons
Poly A sequence: end recognition sequence

Question 17

general process of pronuclear injection

Answer 17

1. DNA injection into male pronucleus of fert. egg (before nucleuses merge)
2. fert. egg grows into embryo
3. introduce several embryos into mouse
4. birth
5. somatic cells are tested for presence of gene
6. whichever mice have the presence of the gene are bred to test for the gene in germ cells

Question 18

what are 2 ways you access the germline?

Answer 18

1. change DNA of fert. egg
2. Blastocyst and ESCs by changing/adding cells of ICM

Question 19

What is a pro for pronuclear inj?

Answer 19

Fairly easy to perform
Can examine wide range of things
Wide species range

Question 20

Cons for pronuclear inj?

Answer 20

-Not a lot of control
-CNVs randomly inserted in head/tail arrays
-rearrangements and deletions may occur

Question 21

What is gene targeting in ES cells ?

Answer 21

Involves introducing a DNA construct at a precise site within the genome which allows specific mutations or deletions in the ESC allele
Then, replacement occurs by delivering constructed DNA and selecting for low homologous recombination probability

Question 22

What special factor is needed for gene targeting?

Answer 22

Requires lines of ESCs that can be propagated in culture and also be able to integrate into a developing embryo and differentiate into germ cells

Question 23

Cons of gene targeting

Answer 23

ONLY IN MOUSE

Question 24

Pros of gene targeting

Answer 24

Can replace genes, knock gene out, insert gene
More control

Question 25

Process of gene targeting

Answer 25

1. Introduce a DNA fragment with the altered gene into many ES cells 2. Grow to form colony 3. Test for the rare colony in which the DNA fragment has replace one copy of normal gene 4. Inject ES cells into early embryo after mice have mated and the embryo is retrieved 5. early embryo partly formed from ESC 6. Introduce early embryo into mouse 7. birth 8. somatic cells of offspring is tested for gene 9. these mice are selected and bred to test for gene in germ line cells

Question 26

What is created from gene targeting

Answer 26

chimera

Question 27

What is nuclear transfer?

Answer 27

Cloning, full ESC

Question 28

Process of nuclear transfer

Answer 28

1. Take somatic cell nucleus and remove pronuclei from haploid egg cell 2. inject somatic cell nucleus into the haploid egg cell 3. In vitro oocyte activation/embryo culture 4. Embryo transfer into animal

Question 29

Disadvantages of nuclear transfer/cloning?

Answer 29

-Epigenetic problems - gene expression varies -process is inefficient

Question 30

Advantages to nuclear transfer/cloning?

Answer 30

-Germline access to virtually all mammalian species

Question 31

What is gene editing?

Answer 31

CRISPR/Cas system -Alters a gene rather than adding anything

Question 32

What is CRISPR

Answer 32

clustered regularly interspaced short palindromic repeats

Question 33

what is CAS

Answer 33

CRISPR associated nuclease

Question 34

what does CRISPR-Cas 9 do?

Answer 34

-Alters a gene by injecting: 1. Guide RNA with homologous target sequence 2. CAS9 3. Replacement DNA -Cas9 makes double stranded cut at target region -Either knockout or edits the region by adding double stranded DNA that inserts into the cut

Question 35

Pros of gene editing

Answer 35

1. broad species relevance 2. rapidly evolving and increases in power 3. can possibly create multiple gene modifications at the same time

Question 36

Con of gene editing

Answer 36

1. less efficient for large DNA additions

Question 37

In gene targeting we use _____ and _____ selection

Answer 37

positive and negative

Question 38

Positive selection does what?

Answer 38

Selects for recombinants

Question 39

Negative selection does what ?

Answer 39

ensures the homologous recombination happened

Question 40

what is usually used for positive selection

Answer 40

antibiotic resistance