knockouts Flashcards
Recombinant DNA technology has made it possible to
directly manipulate genes in vivo
Recombinant DNA technology has made it possible to directly manipulate genes in vivo
This allows scientists to learn more about gene function in living organisms.
These approaches enable scientists to create genetically engineered microbes, plants and animals for research and commercial activities (the field of biotechnology).
(to learn more about gene function in these organisms)
The concept is to create a knockout (KO) to disrupt or eliminate ____________ and then ask, _________
copies of a specific gene or genes of interest
“What happens?”
How does one create a knockout mouse?
Creating a KO mouse is labour and cost intensive, but once it is made they can reproduce and often KO mice are shared between researchersCreating a KO mouse is labour and cost intensive, but once it is made they can reproduce and often KO mice are shared between researchers
1.) Design a targeting vector (to be introduced into cells)
- Know the sequence of the target gene
- Know the sequence of the flanking regions
- Integration by homologous recombination, which interrupts or replaces the GOI – thus non-functional protein
- Inserted DNA is often a marker gene (NeoR, GFP, LacZ)
2.) Electroporate into cultured embryonic stem (ES) cells
3.) Introduce into blastocyst by microinjection
4.) Transplant blastocysts into the uterus of a surrogate mother
5.) Give birth to chimaeras – some cells arising from the KO stem cells, others from the natural stem cells from the blastocyst
6.) F1 chimaeras are heterozygous (+/-)
7.) Make sibling crosses to produce mice that are homozygous for the knockout, called null mice (-/-)
8.) KO can result in embryonic lethality when an ‘essential’ gene is disrupted
9.) Conditional KOs – when the KO is activated at a specific time of development
This example uses a marker sequence for neomycin resistance (neoR). Neomy-cin is an antibiotic that blocks protein synthesis in both bacterial and eukaryotic cells. One can select ES cells transformed with the target vector by exposure to neomycin. One could just as easily use the GFP of LacZ genes for the same purpose.
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What can knockout mice be used for?
KO animal models serve key roles in learning about gene function, and are essential for biomedical research on disease genes.
Research findings in KO animal models help us understand disease models in humans.
Some gene KOs can be embryo lethal
Knocking out a gene that is important may kill the mouse (non-viable).
If null mice (-/-) for a particular gene of interest cannot be derived by traditional KO approaches, an alternative approach called a conditional knockout can be used.
Conditional knockouts allow one to
control the particular time in an animal’s development that a target gene is disrupted.
For example, if a target gene displays embryonic lethality, one can use a conditional KO to allow an animal to progress through development and be born before disrupting. Another advantage of conditional KOs is that target genes can also be turned off in a particular tissue or organ instead of the entire animal.
One common method used for making conditional KOs is called the
Cre-lox recombination system.
The Cre-lox system
A common approach to create conditional Kos
Insert loxP sites in the targeting vector, on either end of the GOI
Cross with mice containing the Cre gene (encodes Cre recombinase, which recognises loxP sites and deletes the sequence between the loxP sites
Promoter of the Cre gene is key – tissue-specific or inducible
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Transgenic animals
a.k.a. knock-in animals
