Gene Manipulation Flashcards
What does gene manipulation in vivo involve
What does it mean to interfere with a gene
Can you think of a case when a researcher may want to target a specific gene?
Can you think of a case when a researcher
may want to target a random gene?
What are the ways to interfere with a random gene
What are the ways to interfere with a specific gene
What do you start off with when screening genes
What are the principles of chemical mutagenesis
How does EMS work in random mutagenesis
It is highly effective in creating point mutations
EMS is an alkylating agent that transfers an ethyl group to nucleotide bases in DNA.
EMS ethylates guanine to form O⁶-ethylguanine.
How are drosophila used in genetic screens
What is the life cycle of drosophila
What is genetic screening*
Genetic screening in the lab refers to the process of identifying specific genetic variations, mutations, or markers
How do you look for a mutant phenotype
How can you screen for a suppressor gene
There are ways to automate looking for mutant phenotypes in an organism
Why are drosophila used in genetic screening
Because drosophila has lots of genes (20000) screen and look at genes in one chromosome instead of all of them
How many genes and chromosomes do drosophila have
Ignore meets egg
Ignore screen chromosome part
What is an enhancer phenotype
What are the problems with EMS mutagenesis
What are transposable elements
Transposable elements (TEs) are fragments of DNA that can insert into new chromosomal locations and often make duplicate copies of themselves in the process.
“mobile genetic elements”
• comprise 45% of human chromosomal DNA “middle repetitive DNA”
• contribute to spontaneous mutation, genetic rearrangements, horizontal transfer of genetic material
• aid speciation and genomic change (in bacteria transposons are often associated with antibiotic resistance genes)
• cells must depress transposition to insure genetic stability
How can TEs be inserted into a genome (transposon based random mutagenesis)
What 2 proteins can P elements give rise to and how
What is meant by transgenesis
What is meant by gene targeting
Explain how trangenesis by transposons is done in drosophila
Reporter plasmid - plasmid that has GOI surrounded by seq recognised by helped plasmid (seq is from P element)
Transposase must not be present after plasmids injected otherwise TE will keep moving around
When are the 2 plasmids injected into a developing drosophila embryo in transgenesis*
Syncytial Stage:
At this stage, nuclei are rapidly dividing within a shared cytoplasm, allowing the injected plasmids and transposase to diffuse and target the nuclei efficiently.
The plasmid mixture (reporter + helper plasmid) is injected into the posterior pole of the embryo, where the germ cells will form.
This is done to increase likelihood of successful transgenesis
What would happen if GFP encoding P element was inserted into enhancer elements *
Where do P elements tend to integrate into the genome
5’ UTR
How does the GAL4 UAS system work
What can the GAL4 UAS system be used to do*
Allows the expression of genes in a specific tissue or at a specific time by using a promoter driving GAL4 expression in desired cells or conditions.
Overexpressing or ectopically expressing a gene to study its effects on development, behavior, or physiology.
Ectopically expressed refers to the expression of a gene in a location, tissue, or cell type where it is not normally expressed
How is transgenesis done in mice
Most commonly used method
• Only 5% or less of the treated eggs
become transgenic progeny
• Need to check mouse pups for DNA (by PCR or Southerns), RNA (by northerns or RT-PCR), and protein Loading…(by western or by some specific assay method)
• Expression will vary in transgenic offspring: due to position effect and copy number
How is homologous recombination used to produce transgenic mice*
Difficult to do transgenesis in mammals so do homologous recombination instead
1) GOI inserted between homology arms + selection marker in plasmid
2) The targeting vector is introduced into mouse embryonic stem (ES) cells via electroporation.
The cell’s repair machinery uses homologous recombination to integrate the vector into the genome at the target locus.
3) identify ES cells that have homologously combined with vector and inject these into embryos
4) embryos implanted into surrogate- offspring are chimeric (have normal and modified ES cells)
5) Chimeric mice are bred with wild-type mice to determine if the genetic modification has been incorporated into the germline.
Offspring carrying the genetic modification are identified and further bred to establish a transgenic line.
What are homology arms
Sequences that are identical to regions flanking the target gene in the mouse genome. These guide the vector to the correct genomic location.
What do ZFN, TALEN, CRISPR have in common
What are they used to do
used to make precise changes in the DNA of organisms. They are employed to disrupt, modify, insert, or delete specific genes
Summarise how ZFN works + issues
How does ZNF work (in more detail)*
Zinc Finger Nucleases (ZFNs) are engineered proteins used for precise genome editing
Composed of multiple zinc finger motifs, each recognizing a specific 3-base DNA sequence. Zinc fingers are arranged in tandem to recognize a longer, unique DNA sequence, providing specificity
The zinc fingers of each monomer bind to their respective target sequences on opposite strands of the DNA.
The DNA is cleaved at the spacer site between the two ZFN binding regions.
The cell repairs the break using one of two pathways:
Non-Homologous End Joining (NHEJ) , Homologous Recombination (HR)
How does TALENS work*
are genome-editing tools designed to introduce specific double-strand breaks (DSBs) into DNA at targeted locations
Composed of transcription activator-like effector (TALE) repeats,
Specificity is determined by two hypervariable amino acids (the repeat-variable diresidue, RVD) within each repeat:
NI recognizes A.
HD recognizes C.
NG recognizes T.
NN recognizes G (and sometimes A).
Multiple TALE repeats are assembled in tandem to recognize a specific DNA sequence. (One TALE repeat ,~33 aa long, has one RVD), need multiple TALE repeats to recognise DNA sequence.
Then rest of process same as ZFN (cut at spacer region, repair)
How does CRISPR-Cas9 work*
Components of Cas9 Protein:
A nuclease that cuts DNA, creating DSBs.
Guided to the target sequence by an RNA molecule.
gRNA combines:
- CRISPR RNA (crRNA): Contains a 20-nucleotide sequence complementary to the target DNA.
- Trans-activating CRISPR RNA (tracrRNA): Binds to the Cas9 protein and stabilizes the complex.
sgRNA guides the Cas9 protein to the target DNA sequence through complementary base pairing.
The Cas9 protein binds to the DNA only if a PAM sequence is adjacent to the target site.
Cas9-sgRNA complex binds to the target DNA, and the sgRNA hybridizes with the complementary DNA strand.
This forms an RNA-DNA duplex and opens the DNA double helix.
Then rest of process (ds break, repair via either of the two pathways)
What is the structure of Cas9*
Recognition (REC) Lobe:
Responsible for recognizing and binding to the guide RNA (gRNA) and the target DNA.
Nuclease (NUC) Lobe:
Contains the nuclease activity responsible for DNA cleavage.
Composed of:
HNH Domain:
Cleaves the DNA strand complementary to the guide RNA.
RuvC Domain:
Cleaves the non-complementary DNA strand (opposite to the guide RNA).
PAM-Interacting Domain:
Recognizes the protospacer adjacent motif (PAM) sequence, essential for target DNA binding.
What would happen is you silence the cutting activity of one of the nuclease domains in Cas9
Why would you want to do this
produces only single-strand breaks, reducing the risk of large-scale genomic disruptions
If two single strand nicks are near to eachother = considered a ds break
(Ignore article )
How could you still get dsDNA breaks even when you silence one nuclease domain
What is a PAM sequence in CRISPR*
It is located immediately adjacent to the target DNA sequence that the Cas9 protein will bind and cut. The presence of a PAM is required for the Cas9 protein to recognize and cleave the target DNA
What is meant by homology directed repair of a dsDNA break*
DNA repair mechanism that cells use to fix double-strand breaks (DSBs) in DNA. It relies on the presence of a homologous DNA template to guide the repair process, ensuring accurate restoration of the DNA sequence at the break site. HDR is a critical pathway for maintaining genome stability
What is meant by non homologous end joining repair of a dsDNA break*
is a DNA repair mechanism that fixes double-strand breaks (DSBs) in DNA without requiring a homologous template. It is an error-prone but fast and efficient pathway
lack of a template often leads to mutations (indels) at the repair site which can disrupt gene function by introducing frameshift mutations or premature stop codons.
How is CRISPR used in bacteria to defend itself against bacteriophages *
The system helps bacteria recognize and neutralize these viral threats by “remembering” previous infections through a form of genetic memory and using it to target and cut the phage DNA during subsequent infections
When a bacterium is infected by a bacteriophage, the CRISPR-Cas system can capture a short segment of the viral DNA, called a spacer sequence.
This sequence is inserted into the bacterial CRISPR array, which is a repetitive DNA region in the bacterial genome.
Each new spacer sequence represents a “memory” of the virus that the bacteria has encountered
CRISPR array is transcribed into a long RNA molecule, which is then processed into smaller RNA fragments known as crRNAs.
Each crRNA contains a sequence (called the spacer sequence) that is complementary to a corresponding segment of the viral genome the bacterium has previously encountered
crRNA guides the Cas proteins (e.g., Cas9, Csm, or Cmr complexes) to the phage DNA.
The Cas proteins, guided by the crRNA, recognize the viral DNA that matches the spacer sequence and bind to it.
Once the Cas proteins bind to the phage DNA, they cut the viral DNA, rendering it inactive
How is the Cas9 complex and gRNA formed (with reference to tracrRNA) in bacteria*
crRNA is transcribed from the bacterial CRISPR array, which consists of repetitive sequences interspersed with unique sequences (spacers) that represent viral DNA or other foreign DNA that the bacteria have previously encountered.
A crRNA molecule contains a spacer sequence that is complementary to the target DNA sequence (usually within a bacteriophage genome).
tracrRNA is complementary to the repeat sequence of the crRNA, and it helps bind the crRNA to Cas9.
TracrRNA also facilitates the maturation of crRNA by helping to process the crRNA into smaller functional units.
The tracrRNA binds to the repeat region of the crRNA, forming a duplex.
This crRNA-tracrRNA complex then associates with the Cas9 protein.
Cas9 is a two-domain protein that requires the binding of this RNA complex for activation. The crRNA guides Cas9 to the target DNA by base-pairing with the complementary DNA sequence, and the tracrRNA helps stabilize this interaction.
The Cas9-gRNA complex (which includes both crRNA and tracrRNA) scans the DNA for a matching sequence to the crRNA spacer.
Once a match is found, the PAM (Protospacer Adjacent Motif) sequence is recognized by Cas9, and the protein creates a double-strand break (DSB) in the DNA
What is meant by these terms:
What are the different types of CRISPR
What is the timeline of RNAi history
What kind of activity is transcription and translation*
refer to processes or functions that are relatively stable, consistent, and not dynamically regulated in response to immediate changes in the environment or cellular conditions
What did izant and weintraub do
Investigated potential of antisense DNA strand transcription to inhibit gene activity
By flipping the orientation of a gene (the HSV thymidine kinase, or TK gene) and inserting it into a vector, antisense RNA is produced. When this flipped gene is co-injected with the wild-type TK gene at a 100:1 ratio into TK-deficient mouse cells, there is a significant reduction in TK expression and cell viability compared to controls. The reduction is likely due to trans-inhibition by antisense RNA, not artifacts from DNA rearrangement. This demonstrates the potential of antisense approaches for gene regulation.
Give examples of plant genomes that were manipulated
What were the results from the investigation done on genetic interference by ds RNA in C elegans
What two approaches are used to identify molecules involved in RNAi
What are the two homologues of DICER
What is RNAi
A process in which small RNA molecules regulate gene expression by silencing specific mRNAs, effectively reducing or preventing the production of proteins.
How can RNAi be used to manipulate genes
by selectively silencing or downregulating gene expression
Works by introducing RNA molecules into a cell, that match the target gene’s mRNA, leading to its degradation or translational repression
E.g. Synthetic siRNAs (small interfering RNAs)
Can be introduced by a vector
Explain the siRNA pathway of RNAi
Presence of ds RNA initiates the process, either endo or exogenous source of dsRNA
DICER cleaves the ds RNA into 21bp long fragments
siRNA loaded onto RISC complex (unwound, one strand binds Argonaute = guide strand, other strand degraded)
The guide strand in RISC directs the complex to complementary mRNA molecules.
Base pairing occurs between the guide strand and the target mRNA
Depending on the degree of complementarity, mRNA silencing occurs via:
Cleavage (Degradation): If the siRNA is fully complementary to the target mRNA, Argonaute cleaves the mRNA at a specific site.
Translational Repression: If there is partial complementarity, translation of the mRNA is inhibited (less common in siRNA-mediated pathways).
Explain the miRNA pathway of RNAi
miRNAs are transcribed by RNA polymerase II (or III) as part of longer primary miRNA transcripts (pri-miRNAs) that often include hairpin structures.
pri-miRNA is processed by the Drosha enzyme into a precursor miRNA, a ~70-nucleotide hairpin structure
The Exportin-5/Ran-GTP complex transports the pre-miRNA from the nucleus to the cytoplasm.
In the cytoplasm, Dicer, an RNase III enzyme, cleaves the pre-miRNA into a 21 nucleotide double-stranded RNA molecule, which is loaded onto RISC
one strand of miRNA binds argonaute= guide RNA, other degraded
miRNAs guide the RISC to target mRNAs through complementary base pairing
degree of complementarity determines the silencing mechanism: perfect complementary = mRNA degraded, mismatch = translation inhibited.
What are the two main categories of RNA silencing mechanisms
What are the different mechanisms of translation inhibition
What is circularisation of mRNA
mRNA translated in a loop (mRNA is in a loop shape)
Significance:
The proximity of the 5’ cap and 3’ poly(A) tail facilitates ribosome recycling. Ribosomes that finish translation at the stop codon can quickly reinitiate at the start codon.
What are the different types of small RNAs
How is mRNA silencing amplified to progeny in C elegans
What is the difference between knock in and knock out and knock down of a gene
Knock-in adds or replaces genetic sequences to study specific modifications or introduce new functionality.
Knock-out completely removes or inactivates a gene to study its absence.
Knock-down reduces gene expression temporarily to study partial loss-of-function effects.
How can you use RNAi to screen genes with reference to multiplate wells
Each well in multiplate contains cells to be treated with a specific RNAi molecule
Use RNAi libraries to target specific mRNAs corresponding to the genes to be silenced.
What different phenotypes have been screened using RNAi
How can Sugar rich vs protein rich food preference in drosophila be studied
They switch cuz lay lots of embryos = energetically costly for female
How can GAL4-UAS system be used for directed gene expression with reference to RNAi
Can be used to :
Knock down genes in specific tissues or cell types
Study gene function at specific stages of development
Perform large-scale RNAi screens to identify genes involved in specific biological processes or pathways.
Knock down genes associated with human diseases in specific tissues to study their effects.
