Experimental techniques in dev biology Flashcards
(9 cards)
painting
visualise when and where genes are expressed
cutting
find out where the gene is needed in the tissues where it is expressed
pasting/transplantation
find out whether the gene alone is sufficient to control the development of tissue
epigenetic must be considered alongside to genetic processes
cellular competence of cells/stem cells
screening by phenotype
- forward genetic screening (from mutant phenotype to gene)
genome wide screen
others
identify genetic modifiers of known mutations
- to Introduce mutations into the genome one must change the DNA - random mutagenesis
how?
exposure to mutagens by chemicals, usually radiation = sizeable deletion carcinogens, or transposons, viruses such as retroviruses.
use of ENU alkalising agent acting to transfers ester group to nucleases in nucleic acids creating AT to CG transitions /1-2 mega base intervals specifically targeting sperm - propagation - interbreeding to generate homozygous
zebrafish have external fertilisation mechanism and 100s of eggs played in one go
let f1 make an f2 generation in separate tanks
mate f2 males with f2 females = f3 generation
according to mendelian genetics
expected that 1/16 animals in f3 generation to be homozygous for the mutation
if the mutation was in an important control gene phenotypes should appear - can identify the gene
- mutant phenotypes and mutated genes identified
problem with genetic screens
must judge how many genes are in the genome
money needed for matings
saturation mutagenesis - setting up a screen where statistically every gene is hit - only will lead to identifying the phenotypes if there is no genetic redundancy
in vertebrates there is because 2 then 3 whole genome duplications occur in succession = leads to redepolidisation through gene loss
genes can still step in when one of the paradox is lost damaged or in this screen mutated = prevents a mutant phenotype from occurring
may want to employ another type of screen
screening by similarity
humans = dueterestome drosophila = protostome
gastrulation is shared
in bilateral there is a tendency towards cephalisation - concentrating the sense organs and the brain at the head end
limbs occur in certain places along body axis
is there a conserved mechanism that specifies positions along the body axis and defines the head?
how can we find if these animals share developmental control genes?
similarity screening
to identify similar protein sequences, antibodies against a conserved part of the protein is needed o identify similar DNA sequences
to identify similar DNA sequences - exploit that nucleic acids can form base pairs via hydrogen bonds = binding where sequences are complementary
cheaper = easier = used more widely
however the genetic code is degenerate
the same amino acid can be encoded by several codons
extreme cases - leucine and Argentine
= DNA can have a divergent sequence and may still code the same protein - search for DNA sequences that are a variation of the same coding sequence
- colony hybridisation
- pcr screen with degrease primers
an approach to identify new genes by means of sequence similarity with known genes
exploiting ability of similar strands of nucleic acids to base pair and form hybrids
a probe can fish out similar molecules from a genomic or cDNA library
on a computer
BLAST
search genomes or CDNA database w the sequence of a known gene
query sequence = virtual equivalent of a probe
screening by expression
- microarray analysis for differential expression - extract RNA and generate cDNA libraries for the control
dna fragments represent all known genes on microscope slides
exploit base pairs of nucleic acid
A/generate probes depend on what you want to find out
i.e. genes expressed at onset of heart development
isolate RNA from one tissue then from another
generate probes for each RNA molecule isolate by reverse transcribing RNA into cDNA in presence of fluorescenctly labelled nucelotides
one cDNA collection is made to fluoresce on colour n the other another
mix both cDNA preparations at equal concentraion
= hybrids them to the dan on the microarray
wash away unbound material
use a laser at a wavelength that excites the fluorochromes
can identify how much of each colour fluorescent material got bound to the dNA from a specific gene
diff colour of diff intensity
black = uno exp
red - unregulated under exp conditions
green - highly exp under original conditions = down regulated under exp conditions
setback
if u want to find new gene - only find what we already know and have anchored onto the array
original arrays become dated
only know he DNA known at the time
less bias way = RNA seq analysis
all RNA form a tissue is converted to cDNA and sequenced by high throughput sequencing
3 biological explicate s required
primary sequences are added that serve as a unique identifier for library then put through high throuput - molecules are anchored to be read and copied by DNA polymerase in a PCR
each newly incorporated nucleotide wither because it carries a florochrome or because it creates an electrical signal will be recorded - raw reads
bioinformatics
filter raw reads + contamination from humans executing experiment
quantification of deregulatioed genes and gene groups
and extent of gene deregulation
microarray or RNA seq screen for genes w specific expression patterns
microoarray analyses cannot deliver novel genes
rna seq analyses can deliver expression data for novel genes identifies in the most recent editions of genomes
both contain reverse transcription and application which introduced error = expression of newly identified gens has to be validates by conventional methods = insitu hybridisation
screening by protein protein interaction
the east 2 hybrid screen is a genetic screen for proteins interacting w the bait
relying on combination of 2 hybrid proteins, the bait fused to the gal4,DNA binding domain, the library of possible targets fused to the gal4 transactivation domain
if bait and target interact = GAL4 domains brought into close proximity = GAL4 sensitive reposer activated
