Manipulations/ biological approaches Flashcards
(27 cards)
What are the major classes/functions of genes?
- Cellular metabolism genes
- Transcription and translation related genes
- DNA replication/modification
- Movement/Corrdination genes
- Basal cellular function
Vast majority of genes identified have unknown function
How can we modify the yeast genome to better understand the function of a specific sequence of the genome?
*Need diploid cell
1. Tranform diploid cell with disruption construct → homologous recombination (need homologous known flanking regions at both ends of the gene) → diploid cell with 1 drug resistant gene and 1 normal gene
- Select for G-418 resistance → cells that did not get the construct can’t grow
- Sporulation → cell division gives 4 haploid cells: 2 with construct, 2 with normal gene
- Put cells without the gene in different environment to see when cell responded differently than normal cell. If get only 2 cells, means that cells without normal gene died → gene was essential for life
How can RNAi be used to learn about gene functions of specific sequences?
By knocking down the gene with RNAi, you see which cell function lacks
Did it with C. elegans for each gene 1 by 1
Introduce plasmid (dsRNA with T7-promotor + IPTG)
How can we use the fact that transcription factors are modular to learn about gene function?
Have a 2-hybrid system (2 fusion proteins):
Bait: DNA binding domain + A-fusion protein
Prey: B-Transcriptional actviating domain + B-fusion protein
Only way to get transcription of a reporter gene is if proteins A and B interact together
What are different types of protein fragment complementation? (Bait and Prey)
Reconstitute a protein, Bait has N-terminuc of the protein and Prey has the C-terminus
- Reconstitue an enzyme ex: dihydrophobic reductase by interaction of protein X and Y → see interaction of protein X and Y, when cell growth is allowed (function of dihydrophobic reductase)
- GFPn + protein X, GFPc + protein Y → when X and Y interact → give fluorescence
How can we analyse indirect interaction between proteins in a cell?
Do Bio-ID by ith proximity labelling
1. BioID-bait plasmid → Bait gene (protein of interest) + BirA* (Biotin ligase → biotinylates)
2. Put Biotin into environment (tags protein that come close to the protein of interest → proximity dependent biotinylation
3. Affinity chromatography → Isolate biotinylated proteins with antibody
4. Run mass spec to identify them
What type of interaction is analysed with the Bait and Prey method?
Direct interaction
What is special about Apolipoprotein B-48?
It escapes NMD (nonsense mediated decay) even if it has a premature stop codon, it is not degraded
What molecule is used for wash off of Biotin after affinity chromatography to isolate biotinylated proteins in Bio-ID proximity labelling?
What are new variation included?
Avidin binds with higher affinity to Biotin than tagged proteins → can wash off to collect your proteins of interest
What are new Bio-ID proximity labelling technics other than Biotin labelling?
- APEX and APEX2
- TurboID
What is reverse and forward genetic analysis?
Forward genetic analysis: Disrupt homeostasis based on random mutations
Reverse genetic analysis: Disrupt the activity of specific gene product to assess its function (look at new phenotype)
How can homologous recombination be performed in pluripotent stem cells of mouse?
Formation of ES (embryonic stem) cells carrying a knockout mutation:
1. Make a Gene X replacement construct: HSV-tk sequence + flanking regions at both ends of G-418 resistant sequence:
- Introduce construct into cell → 2 possible outcomes:
- Homologous recombination (with flanking regions) → replaces the target gene you want to knock out → only G-418 introduced in ES-cell DNA
- Nonhomologous recombination → neo r (g-418 resistant region) and HSV-tk introduced in DNA as a random insertion - Treat with G-418 resistant (all recombination events, will eliminate only cells that did not do recombination at all) → positive selection
- Treat with Ganciclovir → will kill cells with HSV-tk sequence (random insertion) and not the one that did homologous recombination → negative selection
You end up with cells that have a specific knock out gene / targeted disruption in gene X
What is the particularity of HSV-tk gene?
HSV-tk inserted into cells → phosphorylates ganciclovir → toxic to cells
How can homologous recombination be performed in totipotent stem cells of mouse?
- 4.5 days blastocyst of a/a, X+/X+ recessive black mouse
- Inject ES cells dominant A/A, X-/X+ brown mouse
- Tranfer embryos into psuedopregneant femal (foster mother) have heterogenous population of totipotent cells in blastocyst
- Get mixture of both genotypes:
- Black mouse
- Chimeric (2 colors)
Recombination will be passed down in the germ line!
What is the difference between a pluripotent and totipotent stem cell?
Totipotent:
- Most potent and versatile type of stem cells
- Found in very early stages of embryonic development
Pluripotent:
- Very versatile, but can’t differentiate into extraembryonic tissues like placenta
- Found in later stages of embryonnic development
How are transgenic mice made?
- Inject foreign DNA into on of the pronuclei → fertilize the mouse egg prior to fusion of mal and female pronuclei
- Transfect injected eggs into a foster mother (mouse) → ~10-30% of mouse will contain foreign DNA in their chromosomes of all their tissues and germ line
- Breed mice expressing foreign DNA to propagate DNA in germ line
Which genes are important for understanding expression patterns of genes?
How can they be expressed?
Transgenic reporter genes
Can be expressed under:
- endogenous promotor (from gene that is normally expressed)
- heterologous control (regulatory sequence that is NOT native to the organism in which it is introduced)
*They can be used to edit the genome (of transgenic mice for example)
What characteristic of bacteria allowed the discovery of CRISPR?
- Segment of bacteriophage (type of virus) DNA are integrated in some bacterias in CRISPR (palindromic regions)
- Regions are transcribed into primary RNA that is bound by tracr/trRNA
*tracr = trans-activating CRISPR RNA, trRNA = transfer RNA - Cas9 recognises structures in tracrRNA and is recruited to foreign DNA segments that are recognized by CRISPR RNA
What does CRIPSR stand for?
Clustered Regularly Interspaced Short Palindromic Regions
Short Palindromic regions = repeats regions of same sequence
Interspaced with specific DNA sequences that correspond to target sequence (ex:Bacteriaphage DNA you want to kill)
What is Cas9 and its structure?
Has 2 endonucleolytic cleavage domains :
- RuvC domain → cleaves the non-target strand
- HNH domain → cleaves the target strand of DNA
Both at specific site 3nt uptstream (5’) of the PAM sequence in target DNA
Has sgRNA binding domain
Has PAM recognition domain
Has C-terminus domain
*Cas9 has no specificity on its own
What is an sgRNA?
- Engineered combination of bacterial crRNA (corresponding to target gene) + tracrRNA (stabilizes cas9)
- Has complementary to target DNA sequence
- Will recruit Cas9 to the target region of genome
What is PAM and its importance for CRIPSR?
PAM = Protospacer Adjacent Motif (NGG) short conserved DNA sequence
Specifically recognized by Cas9 for binding to target DNA and initiating cleavage
- Adjacent to target DNA sequence recognized by sgRNA
- N(any nT) + GG, choose Cas9 according to the PAM sequence you chose in the DNA downstream from the target gene
How do we make both Cas9 and sgRNA present in a cellas they are not?
Using a trangenes, both in same cell nuclei:
- sgRNA gene = Promotor + Target sequence (crRNA part) + RNA scaffold (tracrRNA part)
- Cas9 gene: Cas9 coding sequence + NLS
Then sgRNA will be recognized and bound by Cas9 → brought to target DNA site bc of sgRNA and PAM specificity
How does the cell respond to a double-strand break caused by Cas9?
- Does Nonhomologou end joining → short deletions, mutations → disrupts open reading frame (ex: could induce a premature stop codon the mRNA that’ll be transcribed from that DNA sequence)
