L5 - Advance Techniques Flashcards
What characterises developing cells?
TF & cell surface molecule expression
Gene discovery
comparison of differentially expressed genes
other main gene discovery route
screening/cloning approaches in normal vs diseased
Techniques to identify gene expression
in situ hybridisation
immunohistochemistry
How many markers can be studied in in situ hybridisation/immunohistochemistry
3
multiplex in situ hybridisation
hybridisation with multiple probes
Bioinformatics use - developmental
hierarchically cluster genes into spatial groups
Technique to read entire transcriptome
gene annotation coupled with single cell RNA sequencing
Single cell gene expression allows
identify different cell types
determine heterogeneity
find new cell surface markers
Function of combination of approaches
higher spatial resolution of genes
Overlap of spatial expression
regulatory relationship or common lineage between two lines
Transcription dependence
activator binding to specific enhancer
Transgenic reporter lines
transgenic animal with fluorescence component downstream of enhancer
Constitutive knockout
target gene is completely inactivated in every cell
Problem with constitutive knockouts
embryonic lethality
Inducible KO model advantage
bypasses problems associated with constitutive knockouts
inducible KO model
gene temporally suppressed at specific time point
cre
cuts at lox sites
recombines ends
Cre-lox system depends on
cre-line that drives expression of gene in defined tissue or cell
Cre-lox system creates
temporal conditional knockout mouse models
lineage tracing techniques
cre-lox system + GFP / marker
Optogenetics
individual neurons activated by specific wave of light
