1) Intro/Tech Flashcards
The phylotypic stage
The most conserved stage of development between species within a phylum. A period of maximal similarity between species within each animal phylum.
Cause of death prior to E8
preimplantation loss
- disruption of basic cellular functions
- no blastocyst formed
peri-implantation failure
- no connection between trophoblast and the decidua
death at gastrulation
- failure to form the definitive germ layers
- failure to elaborate mesoderm
Death during organogenesis (~E10-14)
- blood flow in the yolk sac
- failure to establish a chorio-allantoic placenta
Death in early fetal period (~E14-19)
- heart defects
- liver hematopoiesis
Pros of Transgenic mice
- simple to make construct
* easy to obtain transgenic mice/cells
Cons of Transgenic mice
• cannot control genomic location of insert
• position effect = variability of expression, sometimes silencing • often multiple insertions, or integration of transgene array
(sometimes > 100 copies)
Goals of Gene targeting and chromosome engineering in the mouse genome
Test gene function in vivo in a mammalian model system
Reverse genetics in mammals : what is the function of my gene?
Tissue-specific loss and gain of function mutations
Model human disease
• single gene mutation, deletions or point mutations
• multiple gene disease
• aneuploidy
• chromosome abnormalities
(deletions, insertions, inversions, translocations)
Gene targeting and chromosome engineering in the mouse genome
- Build a targeting vector specific to your favourite gene.
- Produce a mutation in ES cells by electroporating the construct that will target a change to one allele of this gene following homologous recombination.
- Isolate clonal lines of correctly targeted ES cells > confirm mutation.
- Produce ES cell chimeras by injecting the correctly targeted ES cells into a host mouse blastocyst or by doing a morula aggregation.
- Breed the male chimeras to recover offspring carrying the targeted mutation through germ line transmission of the ES genome.
- Analyse the results of the mutations in heterozygous and homozygous mice by breeding: phenotypic analysis.
Requirements of a targeting Vector
- homologous DNA: ideally from same mouse strain as ES cells… library screening
genomic PCR : easy with genome sequence available! 2. positive selection for vector incorporation
(neo, puro, hyg, zeo,…) - negative selection against random integration events
(tk, DTA; facultative) - screening strategy for homologous recombination events Southern blot analysis
Genomic PCR
Conditional mutations
For conditional mutagenesis, two different constructs must be brought into the same mouse by breeding: the conditional allele (loxP target) and the tissue-specific Cre line
This mouse can be homozygous for the conditional allele, or preferably heterozygous (null allele/conditional allele; KO/cKO).
That way, the Cre excision can more efficiently generate null/null (KO/KO) cells.
The resulting mouse is a mosaic: null/null cells are only found in Cre-expressing cells.
How does CRISPR work?
How does it all work?
• tracrRNA + crRNA form a complex
• recruits Cas9
• the spacer of the crRNA finds its match, the proto-spacer
• this targets the Cas9 complex
• Cas9 introduces a DSB 3 bp upstream of the PAM sequence (proto-spacer adjacent motif)
What is Cas9?
- bacterial nuclease guided to target DNA site by a small RNA molecule!!
- works as an RNA-guided nuclease in eukaryotic cells
What are 2 ways Cas9-induced double strand breaks can be repaired?
non-homologous end joining (NHEJ)
• error-prone, generates small random deletions/insertions (INDELs)
• very efficient, only requires Cas9 + sgRNA
• with 2 sgRNAs, can be used to generate larger deletions (Mb range)
Homology-directed repair (HDR)
• precise, can be used to introduce a single base pair mutation
• less efficient, requires Cas9 + sgRNA + fragment of homology
What is a conditional mutation?
WT in permissive environment, MT in restrictive enviornment
Are lines for conditional mutations
Cre lines generated by transgender or KI alleles (specific to mice)
