lecture 5- methods & microscopy Flashcards
what is pronuclear microinjection
DNA is injected in the embryo and taken up into the genome
- initial problem: “positional effect,” DNA integration was random, so different transgenic often had different expression due to their position in the genome
- homologous recombination
what is homologous recombination and what became the problem & solution
- targeting of inserted DNA to a specific site in the genome
- through the exchange of equivalent amount DNA from organism and foreign plasmid
- allows one to remove an entire gene, “knockouts”
- insertion of another gene “knock in” –> lethality became problem
solution was conditional knockouts via the CRE-LOX system
describe homologous recombination steps
identify gene u want to knockout (YFG)
- generate a plasmid or other DNA vehicle that contains an empty region that is flanked by border sequence identical to YFG
–> put plasmid in with genomic DNA often in embryo –> matching flank sequences will align & recombination will occur
–> now u have targeted and eliminated YFG from your genome
describe conditional/inducible homologous recombination using CRE-LOX
lox sites are 34b of sequence from bacteria phage, these can be placed into a genome (flank a gene)
- CRE is a specific site recombinase that excises material in between LOX sites
in homologous recombination with CRE-LOX, f you express CRE with a cell or tissue or now an inducible promoter, then…
then you can cause a knockout in a specific cell or tissue under your control
for homologous recombination, why might conditional or inducible be better?
you control the effects
- temporally- if a gene if required in the embryo, then you can now see what it is doing later on in development, by not knocking it out until after the embryo is formed
- specific conditions- certain stresses or after infections
spatially- what happens in one part of the organism
name advantage and limitation of CRISPR
advantage- targeted gene editing
limitation- still requires transformation; off site (extra) targeting sites
new: Cas13 - RNA editing and Cpf1 - different cut and editing sites
name 5 methods of transcript analysis (RNA expression analysis)
1- northern blot
2- RT-PCR
3- Realtime PCR (qPCR or qRT-PCR)
4- microarray
5- RNA-seq
describe method of northern blot
RNA is isolated, run out on a gel to give a size gradient spread and transferred to a nylon membrane –> DNA of choice is used as the probe (radioactive) that will anneal to complementary RNA
in northern blot, the amount of signal = __
the amount of RNA or gene message present
in northern blot, running a gel (electrophoresis), it separates by ___ using ___
size
charge
PCR-based RNA analysis methods
reverse transcriptase (RT-PCR) vs. real-time or quantitative PCR (qRT-PCR)
- both PCR based methods to determine gene expression levels in samples
- RNA is converted back to DNA (cDNA) on which PCR can be conducted
- amount of PCR product depends on original amount of RNA present (transcript or gene signal)
PCR is a method to ___/___ DNA
copy
amplify
describe RT-PCR (reverse transcriptase)
extracted RNA is converted back to DNA (cDNA) on which PCR can be done in a semi-quantitative manner
- amt of PCR product depends on original amt of RNA present (** this can only be observed by ending the PCR rxn**)
describe real time (qRT-PCR or qPCR)
a fluorescent dye or labeled primer is added to the PCR rxn of cDNA from a RNA sample and then a laser monitors the amt of fluorescence made at every PCR cycle
