Lecture 4: Generation and use of transgenic mouse Flashcards
What is a transgenic mouse?
A mouse that contains additional, foreign DNA
Transgenic mice are used as ways to modify the genome, describe some modifications?
- Delete a specific gene
- Ectopically (in tissues where they are not normally expressed) over-express a specific gene
- Monitor gene expression
- Genetic changes can be permanent/inducible/reversible/whole animal or tissue-specific
What are the three main routes to generating transgenics?
- Pro-nuclear injection: injecting DNA to the pro-nucleus of the fertilized zygote (easiest way)
- ES cell implantation into the blastocyst
- Genome engineering (CRISPR-Cas9)
- Other means of gene editing exist: siRNA (cause the degradation of messenger RNA), ZFNs, TALENs
Embryonic stem cells (ES cells or ESCs) are..
…pluripotent stem cells derived from the inner cell mass of a blastocyst
Describe the process of Pronuclear injection of DNA
- Zygote held in place under the microscope by holding capillary with suction
- A fine glass capillary needle filled with DNA is injected into the male (because larger than the females) pronucleus
- Some of that DNA will get incorporated at random in to the genomic DNA of the mouse
- The zygote is then grown in the lab
- When it reaches a blastocyst stage it can be implanted into an animal, off springs of which are transgenic animals
- Screen the offspring to see which ones have taken up DNA
Are ICM cells pluripotent? What does that mean? How can it be used to generate transgenic species?
The ICM cells contribute to embryo whereas the trophectoderm (trophoblast) wont. They are pluripotent which means they can give rise to all tissues in adult animals
- If ICM are taken form the donor blastocyst and injected into the host blastocyst
- Result: integration of some of the ICM cell from the donor into the embryo derived from the host à Chimeras (a mixture of genetic contributions forms 2 different strains)
- If the chimeric animal and the host animal are crossed, we get segregation of those genes
- We hope for some of the ICM have contributed to the gonads of the chimeric mouse
How can ICM cells be a source of ES cells? How are they maintained?
ES cells are isolated from the ICM
- ES cells can be maintained (grown indefinitely) in culture
- established cell lines exist
Grown on a feeder layer of mouse embryonic fibroblasts (MEF), or on gelatin
- MEFs mitotically inactivated, but metabolically active so they release growth factors and ECM molecules
- This is supplemented with foetal calf serum which provides even more growth factors + LIF (Leukemia inhibitory factor) which helps maintain pluripotency
Properly maintained ES cell cultures retain their pluripotency
Describe the Genetic modification of ES cells? How do they contribute randomly to the embryo? How is Germline contribution determined?
As we grow the ES in culture (above question) we can genetically modify them
- GOI in a plasmid construct added to ES
- Pick the ones that took it up
- Inject them into a host blastocyst
- In the first round we end up with chimeric animal
- We breed that back with the host WT à genes will then segregate à heterozygote animals that are crossed to give à 25% homozygote animal, 50% heterozygote and 25% WT
The above techniques apply to random integration of genes into the genome but often we prefer to integrate the DNA into a specific location into the genome this is done via gene targeting method that replies on homologous recombination
Describe a process of Gene targeting in contrast to random integration?
- The target gene is what we want to modify
- A target vector homologous to target gene is made
- The homologous DNA is mixed with genomic DNA we want to modify, at some low frequency we get homologous recombination.
- Neo gene confers resistance to antibiotic called G418
- Because the homologous recombination is a rare event we positively select the ES cells using the antibiotic
The presence of the blue HSV-tk is to guard against random integration events
- Random integration in the genome might also occur therefore negative selection of HSV-tk in ganciclovir is done
Because homologous recombination is rare, we need to be able to select the ES cells in which it has taken place. Describe 2 selection strategies?
Positive selection: include a gene in the recombined region that confers resistance of those cells to a toxic drug
- Neomycin phosphotransferase (neo)
- Makes cells resistant to G418
Negative selection: include a gene in the construct that confers sensitivity to a toxic drug in those cells in which random integration has occurred
- Herpes simplex virus thymidine kinase
- makes cells sensitive to ganciclovir or FIAU
What can be done with random integration of DNA?
DNA injected into the zygote will integrate randomly in the genome
- ectopic/foreign gene expression or over-expression
- we can make Interfering constructs, eg shRNA
- Function of regulatory regions – instead of injecting a coding region of the gene we can inject a regulatory region and see what effects that has on the expression of the gene
DNA transfected into ES cells (nucleus) will also integrate randomly in the genome
- As above
- This property has been used to generate “gene traps”
Draw out an example of a complete transcriptional unit for ectopic random transgenesis?
To express a particular gene ectopically in an animal we need all the below elements
- Coding region
- Transcripition start site so that it can make mRNA
- Start codon and stop codon
- Tailed with intron and PolyA (signals to stop making mRNA in case it integrates in a region where there is another gene it will stop making that other gene as well)
- Regualtory regions: promoter and enhancer – to drive expression of the gene
What different types of promoters can be used?
- Ubiquitous (drive expression in all tissues), Tissue Specific
- Inducible – on and off switches using drugs
- Length – we can change the length of the promoter
What different genes can be present?
We can use genes of different species - Tag: Flag, myc, GFP, beta-galactosidase – so that it can be detected more easily
Testing conserved, non-coding DNA for enhancer activity
Trapping is performed with gene trap vectors whose principal element is a gene trapping cassette consisting of a promoterless reporter gene and/or selectable genetic marker, flanked by an upstream 3’ splice site (splice acceptor; SA) and a downstream transcriptional termination sequence (polyadenylation sequence; polyA).
When inserted into an intron of an expressed gene, the gene trap cassette is transcribed from the endogenous promoter of that gene in the form of a fusion transcript in which the exon(s) upstream of the insertion site is spliced in frame to the reporter/selectable marker gene. Since transcription is terminated prematurely at the inserted polyadenylation site, the processed fusion transcript encodes a truncated and nonfunctional version of the cellular protein and the reporter/selectable marker. Thus, gene traps simultaneously inactivate and report the expression of the trapped gene at the insertion site, and provide a DNA tag (gene trap sequence tag, GTST) for the rapid identification of the disrupted gene
Gene trapping, by contrast, relies on random integration of a promoterless reporter construct (10, 11) and is limited to genes expressed in ES cells. The most widely used vectors contain a splice acceptor and polyadenylation signal flanking the βgeo reporter gene such that the reporter is activated upon insertion into introns of genes
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC149045/
Gene traps (usually in ES cells)
- Promoterless reporter construct containing a splice acceptor and polyadenylation signal flanking the βgeo reporter gene β-geo:* *β-galactosidase (AKA lactase – enyze turns blue in presence of x ) and neo resistance gene (giving us antibiotic resistanmce in G418)
- Random integratation in ES cells occurs
- βgeo reporter gene is activated upon insertion into introns of genes
- Postive selection using G418 used to identify cells expressing gene
- This shows us that the gene has been integrated into an area of the genome that ahs been actively transcribed. Because it doesn thave its own promotrer it must be the endogenous promoter, because it has a SA it has to be near a splice donor
Splice acceptor (SA)
Describe the Cre-loxP system for conditional transgenesis?
- Cre is a bacterial recombinase (enzyme) that recognises segments of DNA called LOX P sites (short sequnence of 34bp – palidromic on sides middle not palidromic which allows the LOXP sirtes to be directional)
- Cre recognises loxP site à binds to it à induce DNA recombination between 2 loxP sites à deletes a region
Cre-lox transgenics
Lets say we want to delete the first exon | Insert loxP sites into the introns that flank the exon to be deleted | The loxP site is a 34bp sequence that is recognised by Cre-recombinase | Mammals don’t have Cre-recombinase
To insert loxP sites, the region of genomic DNA is cloned into bacteria and modified, then used to generate ES cells by homologous recombination | ES cells are used to generate a mouse line with a “floxed” (flanking loxP) allele
These mice are normal | Cross the floxed mouse with a Cre mouse | The Cre mouse is a separate transgenic animal | Expression of Cre can be driven by different methods:
eg knocked in to an existing gene, so controlled by that gene’s promoter
OR
driven by a promoter of its own – this could be expressed in all tissues, or some and expressed all the time, or at specific times
OR
can be turned on/off by specific drugs that are given to the mouse eg tamoxifen (43)
Wherever Cre is expressed, it recognises the loxP sites and Cre-driven recombination removes the floxed exon | Once recombination has occurred, that DNA will be deleted in that cell and all its descendants, for ever! | Deleteoin will produce a phentopye we are interested in
CRISPR variations
