Transgenic and gene targetting technologies PLS FINSIH Flashcards
What are the three categories for identifying a gene of interest?
Transcriptome profiling, protein-based methods and whole genome sequencing.
What are some of the transcriptome profiling methods?
RTase-polymerase chain reaction, DNA microarrays and RNA-seq.
What are two protein based methods to identify a gene of interest?
1- and 2- hybrid screening, immunoprecipitation.
What are in vitro limitations of studying gene function compared to in vivo methods, in terms of cells?
Not all cells/tissue types are able to undergo in vitro culture.
What are in vitro limitations of studying gene function compared to in vivo methods in terms of matrices?
There are limited (heterotypic) matrices and cell interactions.
What are in vitro limitations of studying gene function compared to in vivo methods in terms of factors?
Limited/no interstitial, endocrine and other (circulating) factors.
What are in vitro limitations of studying gene function compared to in vivo methods in terms of developmental modelling?
There is limited/no developmental modelling.
What are in vitro limitations of studying gene function compared to in vivo methods in terms of organism level analysis?
There is none as it’s in vitro - e.g. can’t study learning or response to stress.
What are the steps for manipulating genes in vivo?
Manipulation in bacteria (gene cloning), transgenesis, conventional gene targetting and genome editing.
What does transgenesis allow?
Visualisation of gene expression in the whole animal.
Give examples of fluorescent reporter proteins.
GFP, mCherry.
What is the range of sizes that transgenes can be?
0.5kb to >1000kb.
How can transgenesis be carried out?
Inject the transgene into the 1-cell embryo (pronuclear zygote) and the gene will integrate into the genome.
What is an alternative of injecting a transgene into an embryo?
Microinject the transgene DNA and sperm into an unfertilised oocyte.
What happens in transgenesis when the gene has been injected into the egg?
It is transferred to the mother and the embryo gives rise to the offspring. All cells in the offspring have the transgene.
What is the efficiency of injecting the transgene into the egg to produce transgenic offspring?
10% will be transgenic.
What are most transgenic animals?
Mice.
When were ES cells first reported in mice?
1981.
When were ES cells first reported in humans?
1998.
Where were the ES cells found in the mice?
The inner cell mass of blastocysts.
How can blastocysts be used for gene targeting?
Genes can be injected into blastocysts and contribute to development.
What is the method for using ES cell gene targeting?
Make a targeting vector DNA construct (TV).
What is the selection method for targeting vectors?
Positive-negative selection.
What is required with targeting vectors?
Homology arms and a reporter.
What are homology arms?
Arms either side of the targeted insertion that are 5-5-15kb in length and recombine by homology direction repair (HDR) with the genome.
What is the typical construct for a transgene?
Promoter, gene of interest and then the reporter.
What is a pronucleus?
The nucleus of a sperm or egg cell during fertilisation. Sperm becomes a pronucleus after entering the ovum, but before the genetic material fuses.
What is pronuclear injection?
When the DNA integrates into the genome.
Does transgenesis usually result in loss or gain of function?
Gain of function, as a gene has been gained.
Give an example of positive selection that can be used for the targeting vector.
Neomycin resistance, selecting with G418.
Give an example of negative selection that can be used for the targeting vector.
Thymidine kinase. You can counter select with Ganciclover which is metabolised by thymidine kinase to produce cytotoxin.
What is homology directed repair?
A mechanism in cells to repair double stranded lesions - used for the homology arm mechanism.
What does transfecting mean?
Infecting a cell with free nucleic acid.
What happens when the targeting vector has been successfully selected?
The embryonic stem cells can be transfected with the targeting vector and can be plated in culture flasks - this is hopefully what happens.
What is the theory behind positive-negative selection?
Positive selection identifies those where the DNA is present, and negative selection is used to identify where the DNA has been inserted CORRECTLY. It allows for fine selection.
How does negative selection work in the process of targeting vectors?
If the gene is inserted incorrectly (random integration) the thymidine kinase will still be present and the addition of the ganciclover will create a toxin to disrupt the DNA.
How can false positives be identified in transgenesis?
Genomic PCR.
How can the insertion of the correct gene in the correct location be confirmed in transgenesis?
Whole genome sequencing (WGS).
What happens after the target gene has been successfully inserted into the gene?
The ES cells are injected into the blastocyst (3.5 day embryo).
What happens when the transgene has been inserted into the blastocyst?
It can be injected into a pseudopregnant mother.
What is a pseudopregnant mother?
Where all the signs of pregnancy are exhibited by an organism, but without a foetus. They make good recipients for transgenesis.
How can the correct insertion of the transgene be identified in mice?
If a blastocyst is taken from a black mouse and embryonic cells from a white coat mouse, the offspring will have a mixed coat colour.
What germ layers do embryonic cells contribute to?
They contribute to all germ layers, and sometimes germline cells.
What do ES or iPS exist in different species?
Humans, rats and pigs.
What animals have iPS cells but no ES cells?
There are no ES cells for cattle or pig, and iPS cells don’t contribute to germline in pigs.
What technique can be used if there are no iPS or ES cells?
Embryo genome editing can be used.
What are the key factors in genome editing?
ZFNs, TALENs and Cas9.
What is genome editing?
A way of making specific changes to the DNA of a cell or an organism.
What is the technology underlying genome editing?
Double stranded breaks (dsb) are created at specific places in the genome.
How are breaks introduced in genome editing?
ZFNs or TALEN (zinc finger nuclease and transcription activator-like effector nuclease).
What is ZFN?
Zinc finger nuclease
WHat are tandem TALE repeats fused to?
FokI.
What happens after the double-stranded breaks are induced?
Cell machinery repairs the break.
What is the result of the cell machinery repairing the double stranded break?
There may be imprecise repair by non-homologous end joining or precise repair by homology-direct repair.
What does non-homologous end joining (NHEJ) result in?
Small insertion or deletion.
When was the Cas9 system first reported in mammals?
2013.
Where was the Cas9 system originally from?
Bacteria - S.pyogenes.
What are the components of the Cas9 system?
Clustered regularly interspaced short palindromic repeat (CRISPR) RNA, guide RNA (gRNA), and Cas9 (CRISPR-associated (Cas) helicase nuclease, Cas9.
What is the benefit of Cas9?
It is simple to use, RNA-guided endonuclease.
What is the mechanism behind Cas9-CRISPR?
Cas9 introduces a site-specific double strand break, and the cell repairs the break via one of two pathways - the same for ZFNS and TALENs.
What are the two pathways in which the dsb can be repaired in the Cas9/CRISPR mechanism?
Non-homologous end joining (NHEJ) and homology directed repair (HDR).
How do NHEJ ad HDR compare?
NHEJ is imprecise as the ends are rejoined, inserting or deleting a few nucleotides whereas HDR is precise as a different DNA molecule is used as a repair template.
Where does Cas9/CRISPR work?
In mammalian tissue culture cells and embryos.
How can genome editing be applied to mammals?
A 1-cell embryo zygote can be inected so that all cells in the offspring have the genome edit.
What is a modification of the Cas9 pathway?
Cas9 nickase fused to reverse transcriptase - it makes a nick not a break, which is safer.
What is a modification of guide RNA (gRNA)?
gRNA fused to template for RTase called pegRNA (prime editing gRNA). This is one RNA moelcule. This means there is no need for a HDR or HDR template.
