Chapter 6-Genetically Modified Animals

Question 1

What are GMA

Answer 1

• animals with modified DNA through recombinant DNA technology

Question 2

Applications of GMA

Answer 2

1. Study of gene functions e.g. model organisms
2. Model human diseases
3. Drug discovery and development
4. Commercial exploitation

Question 3

Research applications of Saccharomyces cerevisiae (yeast)

Answer 3

Cellular processes such as mitosis and protein secretion

Question 4

Research applications of drosophila melanogaster

Answer 4

• early gene mapping via linkage studies
• mutant screens to identify genes related to specific functions
• 4 pairs of chromosomes
• e.g. Hox genes in body mapping

Question 5

Research applications of C elegans

Answer 5

• study development of simple nervous systems and aging
• embryogenesis
• 6 pairs of chromosomes

Question 6

Research applications of danio rerio (zebra fish)

Answer 6

• mapping and identifying genes involved in embryogenesis

Question 7

Research applications of mus musculus

Answer 7

• study gene function
• model human diseases
• 20 pairs of chromosomes

Question 8

Research applications of rattus norvegicus

Answer 8

Study of physiology

Question 9

Types of genetic modification

Answer 9

1. Transgenic
2. Knockin
3. Knockout
4. Targeted mutation
5. Conditional knockin, knockout and mutations

Question 10

Define transgenic modification

Answer 10

• foreign DNA from same or different species introduced to host bu random insertion, possibly in multiple sites
• usually results in overexpression

Question 11

Define knockin genes

Answer 11

• targeted insertion of the transgene at a specific locus via homologous recombination

Question 12

Define knockout genes

Answer 12

• removal of part of gene so gene function is disabled via homologous recombination

Question 13

Steps in non-targeted transgenesis of mice

Answer 13

1. Design of vector (restriction sites, promoter, transgene, intron, poly-A tail)
2. Insertion of linearised plasmid into pronuclei of fertilised eggs
3. Implantation into pseudopregnant mice (mating with vasectomised males)
4. Screen transgeneic mice by genotyping (cut off a piece of tail then PCR) (these mice are heterozygous)
5. Mate to generate line of transgenic mice (homozygous)

Question 14

Define homologous recombination

Answer 14

• type of genetic recombination where nucleotide sequences are exchanged between 2 similar or identical pieces of DNA
• often used by cells to repair double strand breaks

Question 15

Components of targeting vector in homologous recombination

Answer 15

1. Homologous arms
2. Selection markers (positive and negative)
   - negative selection marker (e.g. herpes simplex thymidine kinase hTK) is expressed when vector is only randomly integrated; when expressed, negative marker kills the cell
   - positive selection marker usually antibiotic resistance gene

Question 16

What does hTK do

Answer 16

• converts ganciclovir to a toxic chemical that kills cells

Question 17

Steps in gene targeting in ES cells via homologous recombination

Answer 17

1. Cultivation of ES cells from mice blastocysts
2. Transfection of targeting vector in ES cells (select with neomycin)
3. Proliferation of targeted ES cells
4. Targeted ES cells injected into blastocysts (form mosaic with normal cells of the inner cell mass)
5. Blastocysts implanted into surrogate pseudopregnant mice
6. Birth of mosaic mice (maybe by mosaic coat colour, not sure abt germ line)
7. Breed with normal mice to see if gene targeting affected germ line
8. Birth of heterozygous mice that can mate again to produce homozygous mice

Question 18

Problem with gene targeting ES cells process and how can you solve it

Answer 18

• if the knockout gene is required for growth and normal development, then there will be no live births
• need for conditional knockout

Question 19

Characteristics of LoxP sites

Answer 19

• 34bp sequence
• 2 symmetric 13bp sequences flanking 1 8bp sequence (8bp gives directionality)
• orientation of LoxP determines how the genetic material is rearranged with cre recombinase

Question 20

2 systems that control expression of cre-lox

Answer 20

1. Tetracycline-controlled expression system

2. Tamoxifen-inducible Cre

Question 21

Describe the tet-off system

Answer 21

• in the absence of tetracycline/doxycycline, the tetracycline-controlled transactivator protein (tTA) is able to activate the transgene with cre-lox
• presence of DOX makes tTA unable to bind to TRE (tet responsive element) to activate transgene
• promoter for tTA could be tissue-specific

Question 22

Describe tet-on system

Answer 22

• in the absence of DOX, rtTA is unable to bind to TRE and activate transgene
• in the presence of DOX which binds to rtTA, rtTA can bind to TRE/TetO and activate transgene
• allows controlled expression of transgene at specific time and tissue

Question 23

Describe tamoxifen-inducible cre-lox system

Answer 23

• in the absence of tamoxifen, Cre and estrogen receptor (with mutated ligand-binding domain) forms a complex with heat shock protein 90 (HSP90)
• in the presence of tamoxifen, HSP90 dissociates from the complex and tamoxifen binds instead
• new fusion protein translocated to nucleus where it activates Cre

Question 24

Application of Cre recombinase

Answer 24

• lineage tracing (of mammary stem cells)

Question 25

Concerns of CRISPR gene editing

Answer 25

1. Off-target effects
2. Ethical issues such as increasing existing disparities in accessing healthcare, moral objections to the use of embryos in research

Question 26

Steps in CRISPR/Cas9 editing in mice

Answer 26

1. Design guide RNA (if knockout, targeted region should have a PAM sequence: 5’ NGG 3’)
2. Inject mRNA coding for Cas9 and guide RNA (and homologous sequence) into pronuclei of fertilised eggs/ transfect cas gene and sgRNA into ES cells, screen and inject ES cells into blastocyst)
3. Transfer eggs/blastocysts to surrogate pseudopregnant mice
4. Screen mice for desired gene disruption/mutation

Question 27

What is Cre-Lox generally used for?

Answer 27

• most often used to remove a piece of sequence that is flanked by Lox-P sites
• can be expressed in a tissue-specific manner using a tissue-specific promoter

Question 28

What is Cas?

Answer 28

• endonuclease that cuts both strands of DNA at sites specifies by an RNA guide