Differential Gene Expression Flashcards
Differential Gene Expression Postulates
- DNA of all differentiated cells is identical
- With few exceptions the “unused” genes in differentiated cells are not destroyed or mutated; they can potentially be expressed
- only a small percentage of the genome is expressed in each cell, and from that just a portion of synthesized mRNA can be considered “cell-specific” (transcriptome)
Northern blot (mRNA)
RNA isolation, separation and transfer followed by detection with a complementary probe (hybridization)
Reverse transcriptase-mediated PCR (RT-PCR) (mRNA)
RNA is reverse-transcribed first (cDNA), followed by amplification by polymerase chain reaction (PCR) of target DNA
- you can amplify mRNA that have small representation in the tissue
- can design primers for as many targets (unlike norther blot)
- can compare tissues
In situ hybridization (ISH) (mRNA)
identification of target mRNA in tissue by using labeled probes (RNA or cDNA). Allows visualization of gene expression in place (embryo/tissue)
- no need to destroy tissue, just have to stop development of embryo - probes can be labeled with radioactivity, a dye, or a fluorophore
Transcriptome microarray (Gene chips) (mRNA)
detects differential transcription in situations where one does not know which genes will be differentially expressed
- can compare activity of many genes at once from different cell types/conditions/using fluorescent labels
- Excellent throughput
- High specificity
- poor sensitivity
Forward genetics
random mutagenesis followed by a screening
- chemical mutagen (ENU) used to selectively produce point mutations on sperm (AT to TA or AT to CG)
- Screening for a phenotype
- dominant mutations can be found easily (heterozygotes will show a phenotype- gain of function)
- recessive mutations require breeding (phenotype only apparent in homozygotes-loss of function)
- frequently used with simple organisms (fly) but can be used with mice
- don’t need target so its a good place to start when you don’t know where
Reversed genetics/Targeted mutagenesis aka. Gene Knockouts
targeted mutagenesis of a known gene (knockout)
- Determine the action of a known clones gene in a developmental process
- removes a segment of the known gene by homologous recombination (meiosis)
- can be used in both haploid cells, unicellular organisms and complex multicellular and diploid organisms
- can select rare cells where gene got integrated in the same locus
- need target because you need to assign probes
- can do more than knockout like transgenesis (putting gene into another animal)
Transgenesis (modifying DNA)
Like reverse genetics b/c you need to know your target. DNA constructs because you can modify vector to study your gene and you need to put that construct into nucleus of cells to be transcribed.
- expression of mutated genes (active or inactive)
- Increased/decreased expression of a normal gene (dosage effect)
- reporter studies (label cells that express the gene during development) like GFP
Gene editing
fix something on DNA that can be deleterious
CRISPR/CAS9-lates; zinc-finger nucleases, TALENs
RNa interference (RNAi) knockdowns
- DS RNA becomes cleaved by an RNase-type complex in the cytoplasm into 21-23 nt fragments that target homologous mRNA for degradation
- “Dicer” an RNase III enzyme is required for this process in flies, worms and mammals
- Reduces a specific gene product but does not eliminate it totally in most cases
- Can design small interfering (si)RNA to do this or a short hairpin (sh)RNA expressed from a vector
What do you need for transgenesis?
using recombinant DNA methods, build molecules of DNA containing:
- the gene you desire (WT, mutant etc.)
- vector DNA to enable the molecules to be inserted to host genome
- promoter & enhancer sequences to enable the gene to be expressed by host cells
Disadvantages of Transgenesis
- Transgene integration is inefficient:
- random integration into genome, variability in the # of integrated transgender copies, lack of control over gene expression levels
- The transgene is not always expressed: position-effect variation (wherein the chromatin gets integrated)
- founder mice are sometimes mosaic, i.e: they have cells with different genotype so no assurance that offspring will inherit that train
- need to outcross the transgenic mice to wild type mice to obtain stable lines
Applications of transgenesis
- Inappropriate expression or over expression of a gene
- Dominant negative/constitutively active mutant gene expression
- Reporter gene expression for lineage tracing:
- Fluorescent proteins (GFP, YFP)
- Beta-galactosidase (x-gal staining)
- study of transcriptional regulatory elements (enhancers)
To do a knockout what do you need?
- A mapped, genomic clone for the gene of interest
- Make a targeting construct with -6kb of homology arms and selectable markers for positive and negative selection
- A screening strategy involving two probes that allow for the detection of the endogenous locus and the recombinant
Chimera/Mosaic
animal containing cells with different genetic material
