Test2: Lect 13 Tamara Phillips Flashcards
Expression cassette:
Component of vector DNA consisting of a gene and regulatory sequence to be expressed by a transfected cell.
Random Transgenic (knock in) mice: - Define/process:
- Define/process:
1: Embryonic insertion (via pronuclear injection, injection into the nucleus) of plasmid vectors with ability to insert into genome (likely transposase mediated)
2: They either insert your desired gene, or fail to do so. May insert multiple times.
3: note: this does not knockout any native copy of the mouse gene.
Random transgenic (knock-in) mice: - Considerations:
- Considerations: 1: May be expressing only one copy or many copies. 2: insertion could have been in an important region in the genome 3: A marker, like fish, could be utilized to find the insertion site 4: multiple lines usually made, to make sure that the random insertion site is not what is causing your data
Cross-species transgenic mouse: - Difference? - Additional consideration:
- Difference? Same techniques issues and concerns, but the gene you insert is from a different species. Such as a transhuman gene insertion into a mouse. - Additional consideration: 1: Mouse gene promotor and regulatory sequences are used. 2: Measure to see if expression of transgene is equivalent in level to expression of the mouse gene.
How do you create a targeted knock in mouse?
Plasmid instead of inserting randomly inserts my homologous recombination. You provide flanking sequences for desired site of entry.
What would a homologous gene vector look like for knockout or for crosspecies transgenic replacement.
Picture below different insertion options
You can have a reporter for insertion (neo in this mouse) and a reporter for ectopic insertion (insertion of whole DNA of vector at random) marked as Tk+ and outside of the homology flanking sections.
Conditional/tissue specific knockin:
- How can this be done:
- How can this be done:
1: replace an exon with you your mutant of interest performing homologous recombination.
2: loxP/Cre system. LoxP sites flanking your exon or gene of interest are inserted through reciprical recombination so that they replace gene/exon. Cre will cause these loxP sites to cut the gene or exon out. Expressing cre with a tissue specific promotor, or with a an inducible one, allows temporal or tissue specific knockout.
For knock out methods, how are chimeras involved?
Insertion is often done in embryonic stem cells, followed by chimera and hoping for germlines to come from your transgenic background.
Allows you to conform ESCs have mutation before insertion into blastocyst.
In vivo single gene mutation:
- Considerations:
- Considerations:
1: Recombination vectors which are more isogenic have higher targetting effieciency
2: increased length of homologous region increases recombination vectors targetting frequency
3: must be screened, multiple approaches, PCR, southern blot
4: only get mutant if ESCs inserted into blastocyst become part of germline
5: cre excision is fairly slow, and is irreversible
In vivo single gene mutation:-
Advantages:
1: Good for study of gene function and regulation
2: Removes complications associated with pharmacological manipulation (non-specificity)
3: Provides a method for study when no method for manipulating the target protein exists.
- Disadvantages
1: Time consuming, and mutation may be lethal
2: Developmental compensation (unless conditional mutant)
3: Genetic background interactions (may be issue being from human)
4: Not tissue specific (unless tissue specific promoter is used)
5: Cannot study subtle differences in protein, just complete loss
Passenger genes:
- When is this an issue?
- How can you fix it?
- When is this an issue?
1: You have a strain or embryonic stem cell you mutant is easy to make in, but you want to study its effect in another strain of mice. Make mutant, cross breed with other strain, and select for your gene.
2: Issue arrises in that genes nearby will be brought with your mutant gene, and so you cannot tell whether these passenger genes are causing your phenotype, or your mutant gene. - How can you fix it?
1: Don’t mix backgrounds (can be hard to do)
2: Use conditional knockouts (show phenotype only exists when you knockout your gene)
3: Tissue specific and tissue specific conditional knockouts also help
CRISPR:
- Define:
- Uses:
- Define:
Nuclease is guided by ssRNA to make specific double strand break.
- Uses:
NHEJ can lead to some deletions there knocking out gene.
Donor DNA can be ligated into this break, adding DNA.
CRISPR:
- Advantages:
- Disadvantages
- Advantages:
Can use sequence homology as small as 20 nucleotides for ssRNA guide
Fast KO within a month, knock in is a little slower, but still good
Embryonic Stem Cells are not needed
Several fold higher success rate (more mice end up with your mutation)
- Disadvantages
Can have off target affects. Guide RNA is not always a 100% specific for your desired site.
Gene trapping:
- What is it:
- What is it:
1: Randomly insert a gene trapping cassette (promoterless reporter gene and a pol A sequence, which causes truncated mRNA).
2: Screen a lot of mice to see if they have altered phenotype of interest, if they do ->
3: You can use reverse transcriptase to read off 5’ portion of the mRNA, and see what sequence you get.
4: Or you can attack a protein tag into your gene trapping cassette
Gene trapping:
- Advantages:
- Disadvantages:
- Advantages:
1: Rapidly produced, tagged mutations.
2: Genome-wide mutations are possible
3: Reporter expression and sequencing used for ID, rather than phenotypic expression. - Disadvantages:
1: One time event; not all cell-line mutations will transfer into mouse lines.
2: Behavioral screens may work only for major gene effects or multiple traps (you don’t know if you have one trap in a major gene, or multiple traps in mnior genes)?
3: Not really genome-wide; don’t know mutation sites for all genes, and there are “hot spots”.
ENU mutagenesis:
- What is it?
- Phenotype driven vs gene driven:
- What is it?
Agent which causes 1 functional mutation per 1000 alleles tested, producing usually single base pair substitutions.
Mutants of interest found, bred to make f1, f1 crossed back to parental mutant, to make hopefully homozygous individuals with mutant.
Allows genome-wide screening for mutant of interest.
- Phenotype driven vs gene driven:
Phenotype driven: you look for a specific phenotype in your ENU mutagenesis screen. Cumbersome, lot of possibilities.
Genotype driven: You have a gene of interest, and you screen for a particular mutant. Data bases exist of mutants already made, so you can search those and order the frozen sperm
Phenotype driven ENU mutagenesis Screens:
- Advantages:
- Disadvantages:
ENU mutagenesis:
- Advantages:
1: Single gene change independent of position; i.e., randomness with regard to target.
2: Definitive mutant phenotypes present themselves.
3: A phenotypic range of mutant effects: complete loss, partial loss exaggeration of function, dominant negative, or novel gain of function.
4: May identify mutations in novel genes, based on phenotypic change. - Disadvantages:
1: requires a incredible number of mice
2: Hard to find mutant unless in a known gene, because there is no gene marker for what you mutated
3: Multiple mutations might have occured causing the trait not just one.
4: Each mutation may have only had a small effect on phenotype, which will not be noticed by large screen
Posttranslational control of gene expression:
- Examples:
- Why would you use?
- Examples:
siRNA
dCAS9
CRISPRi: CRISPR bound to an inhibitor, inhibit gene expression
CRISPRa: CRISPR bound to an activatory, promote gene expression
- Why would you use?
Depending on delivery method, do not have to modify genome. Potentially finer control of regulation, but it is hard to tell how much of the activity you are inhibiting.
Optogenetics:
- Define:
- Three categories:
- Define:
light activated channels which cause a chain in the cell. These Opsin channels are encoded by a single gene, which can be inserted by a virus. Can be combined with CRE for selective activation.
- Three categories:
- Channelrhodopsins (ChR): conduct cations, depolarize neurons upon blue light illumination.
- Halorhodopsins (HR) conduct Cl ions inward, hyperpolarize upon yellow light illumination.
- OptoXRs are chimeric receptors (rhodopsin-GPCRs) that respond to green (500 nm) light with activation of the biological functions dictated by the intracellular loops used in the hybrid.
Optogenetics:
- Advantages:
- Challenges:
- Advantages:
1: Reversibly manipulate cell activity in the range of milliseconds.
2: Stimulation gone the minute you turn of the light
3: Good spatial resolution, but might not be perfectly directed. Could even lead to back-propagation of excitatory signals. - Challenges:
1: More precise light delivery
2: Bring light reliably deeper into the brain tissue
3: Be able to apply to large brain circuits at multiple sites and multiple layers
4: Suitable light intensity (don’t want photodamage from light)
DREADDS:
- Stands for:
- Are:
- Stands for:
Designer Receptors Exclusively Activated by Designer Drugs
- Are:
Selective GPCRs which are activated by a designer drug which activates nothing else (theoritically). Most common drug is clozapine-N-oxide (CNO), but others exist and more are being designed. They are introduced into cells by viral vectors.
DREADDs:
- Advantages:
- Disadvantages:
- Advantages:
1: can be controlled by CRE if combined with a CRE system.
2: tissue specific systems using CRE are possible. DREADD is placed into gene backwards, CRE recombination reorientates it to face the right way. If CRE is tied to a tissue specific promoter, expression will be tissue specific. - Disadvantages:
1: Temporal properties are controlled by pharmokinetics of their agonists. It may also take differing times for agonists to be degraded accross populations.
2: 30 min - 6 hrs for CNO
3: not as quick as light activtion, and not as easy to stop.
