ANAT 322 Lecture Notes - Lecture 24: Freerunning, Small Interfering Rna, Corticosterone
7 Jun 2018
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ANAT 322 Winter 2017
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Lecture 24: Review
→ Genetic Models in Neuroendocrinology
1. For every technique he mentioned some examples to go along with that and we should focus on the
techniques used, what they are, how to use them but not worry about the specific examples.
2. Transgenic model is not the same as a gene knock-out since you are introducing a foreign piece of
DNA into the host genome and you do not know where it is being integrated (randomly). You can use
this technique for a number of reasons but it is used to overexpress a gene of interest or express a
reporter gene such as GFP or an effector gene like Cre recombinase.
3. For knock-out models you use homologous recombination in embryonic stem cells so you have a wild-
type locus and a targeting construct. Your targeting construct has the selection marker and outside of
that homology region you have a negative selection marker which is important.
Homologous recombination will take place and you will switch out an exon for the selection marker.
You want the negative selection marker there or outside of the homology region because homologous
recombination in embryonic stem cells is relatively rare. If you want to know that it took place
successfully, the negative selection marker should not be there anymore in the recombined construct.
When you end up with the recombined locus in the embryonic stem cell clone, you will inject those cells
into a blastocyst and you will introduce that into a recipient pseudo-pregnant mother. As those
embryonic stem cells start to give rise to all the tissues, you will get a chimeric mouse. You want that
construct that recombined to end up in the germline. The mouse will give rise to different offspring and
you will have some of them that will be wild-type and some of them will be knock-out mice with the
recombined locus.
4. Conditional gene knock-out uses the Cre-LoxP system and conditional means it happens under a given
condition that can be spatial or temporal. It is very site specific so you have two loxP sites and between
them you have one exon and Cre recombinase will catalyse the recombination of the sequence between
those two loxP sites. Basically, you will get the knock-out of that one exon.
It is important that the two loxP sites are not too far apart because the efficacy of the Cre
recombinase system is not always that great so you really want to have only one exon to ensure that
this will happen successfully.
5. Very commonly used is the viral-based delivery of small hairpin RNA constructs and this system can be
used to knock down a gene. This is also more commonly referred to as RNAi technology where the I
stands for interferon. It is a mechanism of post-translational gene silencing.
In order to deliver the RNAi instructions, researchers use viral vectors like the lentivirus, adenovirus,
or adeno-associated virus which are different means of infecting the cell.
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Dna into the host genome and you do not know where it is being integrated (randomly). Your targeting construct has the selection marker and outside of that homology region you have a negative selection marker which is important. Homologous recombination will take place and you will switch out an exon for the selection marker. You want the negative selection marker there or outside of the homology region because homologous recombination in embryonic stem cells is relatively rare. If you want to know that it took place successfully, the negative selection marker should not be there anymore in the recombined construct. When you end up with the recombined locus in the embryonic stem cell clone, you will inject those cells into a blastocyst and you will introduce that into a recipient pseudo-pregnant mother. As those embryonic stem cells start to give rise to all the tissues, you will get a chimeric mouse.
