Chapter 6-Genetically Modified Animals Flashcards
1
Q
What are GMA
A
- animals with modified DNA through recombinant DNA technology
2
Q
Applications of GMA
A
- Study of gene functions e.g. model organisms
- Model human diseases
- Drug discovery and development
- Commercial exploitation
3
Q
Research applications of Saccharomyces cerevisiae (yeast)
A
Cellular processes such as mitosis and protein secretion
4
Q
Research applications of drosophila melanogaster
A
- 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
5
Q
Research applications of C elegans
A
- study development of simple nervous systems and aging
- embryogenesis
- 6 pairs of chromosomes
6
Q
Research applications of danio rerio (zebra fish)
A
- mapping and identifying genes involved in embryogenesis
7
Q
Research applications of mus musculus
A
- study gene function
- model human diseases
- 20 pairs of chromosomes
8
Q
Research applications of rattus norvegicus
A
Study of physiology
9
Q
Types of genetic modification
A
- Transgenic
- Knockin
- Knockout
- Targeted mutation
- Conditional knockin, knockout and mutations
10
Q
Define transgenic modification
A
- foreign DNA from same or different species introduced to host bu random insertion, possibly in multiple sites
- usually results in overexpression
11
Q
Define knockin genes
A
- targeted insertion of the transgene at a specific locus via homologous recombination
12
Q
Define knockout genes
A
- removal of part of gene so gene function is disabled via homologous recombination
13
Q
Steps in non-targeted transgenesis of mice
A
- Design of vector (restriction sites, promoter, transgene, intron, poly-A tail)
- Insertion of linearised plasmid into pronuclei of fertilised eggs
- Implantation into pseudopregnant mice (mating with vasectomised males)
- Screen transgeneic mice by genotyping (cut off a piece of tail then PCR) (these mice are heterozygous)
- Mate to generate line of transgenic mice (homozygous)
14
Q
Define homologous recombination
A
- 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
15
Q
Components of targeting vector in homologous recombination
A
- Homologous arms
- 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
16
Q
What does hTK do
A
- converts ganciclovir to a toxic chemical that kills cells
17
Q
Steps in gene targeting in ES cells via homologous recombination
A
- Cultivation of ES cells from mice blastocysts
- Transfection of targeting vector in ES cells (select with neomycin)
- Proliferation of targeted ES cells
- Targeted ES cells injected into blastocysts (form mosaic with normal cells of the inner cell mass)
- Blastocysts implanted into surrogate pseudopregnant mice
- Birth of mosaic mice (maybe by mosaic coat colour, not sure abt germ line)
- Breed with normal mice to see if gene targeting affected germ line
- Birth of heterozygous mice that can mate again to produce homozygous mice
18
Q
Problem with gene targeting ES cells process and how can you solve it
A
- if the knockout gene is required for growth and normal development, then there will be no live births
- need for conditional knockout
19
Q
Characteristics of LoxP sites
A
- 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
20
Q
2 systems that control expression of cre-lox
A
- Tetracycline-controlled expression system
2. Tamoxifen-inducible Cre
21
Q
Describe the tet-off system
A
- 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
22
Q
Describe tet-on system
A
- 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
23
Q
Describe tamoxifen-inducible cre-lox system
A
- 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
24
Q
Application of Cre recombinase
A
- lineage tracing (of mammary stem cells)
25
Q
Concerns of CRISPR gene editing
A
- Off-target effects
- Ethical issues such as increasing existing disparities in accessing healthcare, moral objections to the use of embryos in research
26
Q
Steps in CRISPR/Cas9 editing in mice
A
- Design guide RNA (if knockout, targeted region should have a PAM sequence: 5’ NGG 3’)
- 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)
- Transfer eggs/blastocysts to surrogate pseudopregnant mice
- Screen mice for desired gene disruption/mutation
27
Q
What is Cre-Lox generally used for?
A
- 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
28
Q
What is Cas?
A
- endonuclease that cuts both strands of DNA at sites specifies by an RNA guide
