Gene Knockout Procedure

[ericjang]

Hi All,

I am having a little bit of difficulty comprehending the procedure for Gene knockout-knock-in. Can somebody explain to me the concept? I have a copy of the Nobel Prize-winning paper "gene modification in mice" which explains this process, but I have a few questions about the process. I would much appreciate it if somebody could verify if my assumptions are correct-thanks!
The Gene knockout process is as follows:

1.) ES cells are transfered to external culture from Mouse blastocyst
My interpretation:
Suppose I wished to produce a species of mouse lacking a certain "A" gene. I extract some embryonic stem cells from any mouse blastocyst (how do I acquire "stem" cells- are all of the cells undifferentiated or do I have to sort through them?) and culture them in a petri dish.
2.) Targeting Vector introduced by electroporation (vector= rare cell carrying gene?)
Here's where I run into my first obstacle. I get that electroporation is used to introduce a targeting "vector", which I assume is the DNA, protein, molecules, etc. that one wishes to add in. The rare cell resulting from electroporation would be any cell that incorporated this foreign factor?
3.) Positive-negative selection
No Idea what this means, but by an illustrated graphic it seems that this is a method to purify the culture until only the rare cell strain is present.
4.) Result= pure population of tareted ES cells
again, I'm not sure how this rare strain was acquired
5.) Targeted ES cells injected into blastocysts, which are injected into foster mothers.
I get the gist of this procedure-the new batch of stem cells are added to a developing blastocyst before any of the original cells differentiate, similar to the first procedure
6.) Birth of Chimeric mice with mutant gene.
How would one identify a chimeric mouse? I can see how a gene affecting physical appearance can be easily identified through fur/body compared to a normal mouse, but what if it was a gene that was not easily shown or takes time to express (e.g. p53)?
7.) Mating with chimeric mouse
The resulting chimeric mouse is mated with a normal mouse to produce some mice expressing the modified gene completely and some without. I do not understand why this is so, why not just breed two chimeric mice together in the first place? And if one parent has no trace of the desired gene, how would how would the gene be expressed in the offspring?
8.) Identify desired progeny with modified gene from undesired progeny.
Again, how would one go about testing if the gene was present or not? And why are the mice non-chimeric?

Overall, this procedure seems more like gene modification instead of knockout- how would a knockout work?

[deleted-2574]

Hi ericjang,

There were a previous post on sciencebuddies on this topic, by Alaa1991 att:

https://www.sciencebuddies.org/science- ... b26#p10105

Does this help?

[ericjang]

Hi davidkallman,

Thank you for this information! So the prerequisites of gene knockout is firstly the understanding of the protein that the gene creates, followed by the precise DNA sequence of the polypeptide chain. Once the sequence is understood, I assume that a synthetic version of the gene is engineered with a defective protein and then introduced into the stem cells? But wouldn't the stem cells reject the foreign DNA? Also, the gene must come with some sort of reporting factor- is GFP added to the gene so that progeny with the new protein will also express the GFP?

That does indeed answer some of my questions- however, I am still confused about the positive-negative selection step where the population is purified to only have the rare cell, and I still do not understand how a chimeric mouse breeding with an ordinary mouse will produce offspring that only express the new traits or the original traits (and no more chimeric offspring).

please let me know if I need to clarify,

thanks,
Eric

[MelissaB]

Eric,

Can you post the entire citation for the paper? I cannot get it at home today, but tomorrow at work I ought to be able to get it and read it and see if I can help you.

I know that with bacteria expressing, say, penicillin resistance as a marker for the knockout gene, a positive selection step would be to add penicillin to the agar plate, so that you only get bacteria that are resistant to penicillin. A negative selection step would probably be similar, only with requiring the knockout gene. (In this case, you would want to have transferred the bacteria to a different agar plate so you could go back and get the bacteria that had the knockout gene from the previous agar plate.)

As for the breeding, without reading the paper I don't understand it either...but perhaps they needed heterozygotes for the allele, and we're both assuming they needed homozygotes? If you have a chimeric mouse, some of its zygotes will be knocked out and some won't, so it will produce, say, O and o alleles. If it breeds with a OO 'normal' mouse, then you would get Oo and OO as offspring. Perhaps that's what they wanted?

I'm not sure how it works in mammals, but I know in bacteria that if you give them a sudden shock--heat, electricity, pH change, etc.--their cell walls become porous and they can take up foreign DNA, which is then sometimes incorporated into the bacterial genome. I'm not sure how it works in mammals, but I presume the process is similar.

[ericjang]

Hi Mellisa,

Thanks! Sorry I forgot to post the article up- I have attached the link to the article below:
http://nobelprize.org/nobel_prizes/medi ... 07/adv.pdf

[MelissaB]

Thanks!

Okay, here is the part of the paragraph that explains the positive-negative selection:

The following year, Capecchi´s positive-negative selection strategy for enriching ES cells containing a targeted disruption of any transfected gene was presented in Nature [37] (Fig 2). A neomycin resistance element (neoR) is introduced into an exon of the replacement vector, which also has a thymidine kinase (HSV-tk) element at its end. Homologous recombination of the targeted gene will result in neoR expression but the tk element will be lost since it was outside of the recombining DNA sequences. In contrast, random integration of the replacement vector will introduce tk as well as neoR into the gene. This strategy was successfully used to disrupt the int-2 gene, which is a member of the fibroblast growth factor (FGF) family [37].

Here's what that means. The DNA put into the cell has a pattern like: neoR_gene_of_interest_tk. If it is randomly integrated into the genome, the whole thing will be integrated, as neoR_gene_of_interest_tk. However, if there is successful recombination of the gene at the desired location, you will only have neoR_gene_of_interest.

Then you have a population of stem cells where you have some in the wrong location (neoR_gene_of_interest_tk) and some in the right location (neoR_gene_of_interest). In this batch of cells, you will also have cells that haven't taken up any DNA at all, so they will not have neoR or tk. The researchers will first do a positive selection step: add neomycin to the cells. This will eliminate any cells that did not take up any DNA, because they will not have neoR. You are then left with a population of cells that took up DNA, but some is in the wrong place (neoR_gene_of_interest_tk) and some is in the right place (neoR_gene_of_interest). Now you 'copy' the plate with the stem cell colonies onto a plate where the cells have to produce thymidine. Only those with the tk gene can do it--which means that some of the colonies won't grow on that new plate. The ones that don't grow are the ones that did not have the tk gene and were therefore incorporated into the right location in the genome. You can then transfer these cells from the old plate to a new plate, culture them, and you have your culture of stem cells.

Does this make sense? These are difficult concepts and I'm not sure I'm explaining them very well; I'm not a geneticist.

[MelissaB]

Sorry, I wanted to post all of that before it timed out on me.

As for the breeding, they said:

An advantage of this scenario is that the first generation chimera will usually be heterozygous for the targeted mutation and that subsequent breeding can be used to generate the homozygous animal. Thus, only one of the two loci need be inactivated, and recessive lethals can be maintained as heterozygotes.

So, you are right in that if they wanted homozygotes, they would need to breed the F1 chimeras together, which would give you about 25% 'normal' mice, 50% heterozygotes, and 25% homozygotes for the knockout gene. The problem is is that many knockout genes are lethal if the mice are homozygotes for them. Thus, they usually want heterozygotes, which they can get by breeding with a 'normal' mouse.

Does this make sense? If it makes you feel any better, I think you already understand more about this process than most college students do!

[ericjang]

Hi Mellisa,

Thanks for the clarification- your explanation was very clear.I understand why heterozygous knockout mice are necessary, but I have a couple questions about the step below:

Code: Select all

Homologous recombination of the targeted gene will result in neoR expression but the tk element will be lost since it was outside of the recombining DNA sequences

I see how the neomycin is paired with the neoR to screen out the cells that haven't taken up DNA, and that the "location" of the neoR-gene-of-interest-tk should remove the tk gene... but I'm not really sure as to how the tk element disappears during homologous recombination. If the tk element is undesired, why is it necessary to add it in the first place?

[MelissaB]

Eric,

Okay. First, do you understand recombination? I've been assuming that you do, but if not, take a look at this: http://en.wikipedia.org/wiki/Genetic_recombination and then let us know if you have more questions.

So, on the chromosomes inside the stem cells before you do anything to them, you would have:

1. Other DNA_recombination point_gene of interest_recombination point_other DNA_other DNA

On the DNA you put into the cells by electroporation, you have:

2. Other DNA_recombination point_neoR_knocked out gene of interest_recombination point_tk_other DNA

So, if you have actual recombination, you will get chromosomes in your stem cells with:

3. Other DNA_recombination point_neoR_knocked out gene of interest_recombination point_other DNA_other DNA

Whereas if you just have the DNA inserted randomly, it will insert the tk as well:

4. Other DNA_recombination point_gene of interest_recombination point_other DNA_recombination point_neoR_knocked out gene of interest_recombination point_tk_other DNA

(Sorry, the example got a bit complicated...)

The presence or absence of tk allows you to distinguish between 3 and 4. You need to be able to do that, because the chromosome in example 4 has a functioning gene of interest--so it won't act like the gene has been knocked out, even though it has a copy of the knockout gene. If you don't separate these two groups, you will end up with only 1 in 1000 mice having the knockout gene, which is a very wasteful way to do an experiment.

Does this make more sense? I'm afraid it's the kind of thing that being able to draw pictures in real-time would really help, but we can't do that here!

[ericjang]

HI Mellisa,

yeah, it makes more sense now

so is my summary of DNA knockout correct?

using a gene library, find out a DNA sequence that will not produce the gene correctly.
Insert new sequence with the following marker genes:
-neo-R vector- screens out cells that have not taken up new DNA.
-tk vector- determines if the new knockout gene is in the right place on the chromosomes.
Insert properly transformed stem cells into mouse blastocysts to create chimeric mouse offspring.
Depending on desired homozygous or heterozygous expression of gene, breed the chimeric mouse with another mouse, either chimeric or original-strain.

[MelissaB]

Yes, it sounds like you understand the process now . I'm glad I was able to help.