Techniques Flashcards
How did we know TFs were there in the past?
- 80s + 90s mostly guesswork from invert. homologs
- Making antibodies against specific purified TFs
- Injected a protein in to a mouse which then makes antibodies so you can extract them and apply them in another host to see the same protein elsewhere
OR
- using mRNA to see where the TF was expressed via complementary DNA bound to a GFP
What was the flaw with studying TFs in the past before modern technology?
- You could only really look at a couple of TFs at a time
What happened after 2015 regarding studying TFs?
- Post-2015 Genomic Advances: Genome sequencing revolutionized transcription factor (TF) research, linking DNA sequences to specific genes, providing vast amounts of data.
- Single-Cell RNA Sequencing (2012): First published in 2012, this technique transformed gene expression analysis by studying individual cells.
Started to be widely used in 2019
What is single cell RNA sequencing?
- Single-cell RNA sequencing identifies and categorizes cell types by analyzing the mRNA expressed in individual cells.
- Cells are isolated in lipid droplets, their mRNA is captured and barcoded, linking each mRNA to its cell of origin
- Bioinformaticians use techniques like principal component analysis to group cells based on their gene expression profiles
What are the benefits and limitations of single-cell gene expression?
+ves:
- Can identify different cell types in the sample
- Can determine heterogeneity
- Can get transcriptomics data for up to 10,000 cells per sample
-ves:
- Provides no spatial information
What is in situ hybridisation?
- Make a complementary strand of mRNA as a flourescent probe and add it to cell to see where mRNA is expressed in a fixed tissue
Limitaiton = until recently you could only look at one TF per sample - doesn’t really paint a picture of transcriptional profile
What is the benefit of RNA scope and hybridisation chain reaction (HCR)
New technique which allows you to examine the spatial relationship of many different progenitors in the sample by looking and multiple mRNA expression in a single sample
What is traditional Fate Mapping?
Inject indelible marker in to cells and you examine where the marker goes
What is the main limitation with immunohistochemistry and in situ hybridisation?
Cannot be conducted on living tissue
What is the solution to the fact that IHC and ISH cannot be done on living tissue?
Reporter lines: Depend on fact that genes are differentially transcribed as a function of the interaction of their promoter/enhancer and the cell specific transcription factors/activators
How do Reporter Lines work?
- The TF that is specific to liver cells (in this case) will bind to the liver-specific enhancer.
- This binding activates the transcription of the GFP gene located downstream of the enhancer.
- The GFP protein is then produced, and its green fluorescence can be detected in liver cells.
How do we look at function?
Knockout/ GOF
What are the issues with early knockout experiments and what is the solution?
Issue: If the gene you’re studying is essential for early development but you’re interested in its role later in development, the animal won’t survive or develop to the stage you want to study.
Solution: Conditional knockout = knockout a gene at a certain time
What are the two methods for a conditional knockout?
- Tissue specific knockout
- Inducible knockout
How does a tissue specific knockout work?
- Mouse 1 (Floxed Gene): A gene of interest is flanked by loxP sites (Flox), enabling targeted removal.
- Mouse 2 (Heart-Specific Cre): Contains heart-specific enhancers driving the expression of Cre recombinase, which acts as molecular scissors.
- Tissue-Specific Knockout: Crossing the mice allows Cre to remove the floxed gene only in heart tissue, leaving it functional in other tissues.
How does an inducible tissue knockout work?
CreERT2 Function: CreERT2 is a drug-inducible version of Cre recombinase, activated only in the presence of tamoxifen, allowing precise temporal control of Cre activity.
Reporter Line Setup: A second transgenic line has a constitutive promoter driving a reporter gene (e.g., lacZ for blue cells or GFP for green fluorescence), but the reporter is blocked by a “stop” sequence.
Controlled Activation: When tamoxifen is given, CreERT2 removes the stop sequence, activating the reporter gene and visually marking the cells.
How can inducible tissue knockouts be used for lineage tracing?
Cre Activation: Cre (or CreERT2 with tamoxifen) removes the stop sequence in specific cells, activating the reporter gene.
Lineage Marking: Activated cells and their descendants express the reporter gene, creating a visible lineage tag.
Permanent Labeling: The reporter remains active in the lineage even after Cre activity stops, enabling long-term tracking.
What are the different KO approaches in different model organisms and why?
Mice - Cre-Lox + tamoxifen
Chick - RCAS retroviral system / CRISPR/Cas9
Zebrafish- Gal4/UAS or CRISPR/Cas9 system
Germline modification is difficult in zebrafish and chicks
In chicks they have to create floxed lines rather than them just being readily available like they are in mice
