Is it hard to make knock-out cells/mice? - (Dec/01/2008 )

is it hard to make knock-out cells?

I have no idea how they make knock-out cells/mice and I really want to learn.
does it need special facility like microinjectors?

do you have to spend a whole PhD project in order to make a knock-out cell? like p53, p21 knock out cells?

can we make multiple knock outs in 1 cell line?

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1. For learning: ES Cell Protocols and Gene Targeting Protocols.

2. Yes you need microinjectors after you have made knockout ES cells. This is best done in a dedicated core facility. Ideally, you also need a dedicated culture room/hood for ES cells work.

3. You can make targeted ES cells within 6 months (from creating a trageting construct, to electroporation, selection of targeted ES clones to confirmation by southern). It will take another 6 months to get the knockout mouse. So, consider a 1 year project for generating the mouse if things go smoothly.

4. Yes, you can make multiple knockouts. You will need constructs with different selection genes for each targeting construct (Neo, Hygro, Puro). Quality of ES cells go down with all additional manipulation, so the best thing is to generate two knockout mice, and then breed them together to get double knockout mice (or more).

There is a lot of technicality and theory to learn. You should get an ES cell targeting handbook from the library.

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thanks CC

so basically when we have double knock-out mice or cell we call it -/-, but if it is only one knock-out mice it is called +/-.

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No.

Wild type ES Cells and Wt mouse are (+/+)

Targeted Knockout ES cells are heterozygous (+/-)

Mice derived from these ES cells are chimera - when further bred, gives you some Heterozygous (+/-) KO mice.

When you breed this Heterozygous (het) KO mouse (+/-) with another het (+/-), 25% of the offspring are Homozygous mice (-/-).

So, a knockout mouse can have one allele of your gene deleted (Het - generally no phenotype), or both alleles deleted (Homozygous).

If you knockout another gene in the same mouse (by breeding with another KO), you get Double KO mouse.

So, if you work with genes A and B

A +/- : Heterozygous mouse (one of the two A alleles deleted)
A -/- : Homozygous mouse (both A alleles deleted)
A +/-, B +/- : Double Het (one of the two A alleles, and one of the two B alleles deleted)
A -/-, B-/- : Double KO (both alleles of both A and B genes are deleted)

If you target the second allele of your gene in these ES cells (or do gene conversion - read more) it becomes Homozygous -/- ES cells.

You have a lot to read. But feel free to ask more questions after a brief reading.

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God, what a complete explanation. you explained better than my supervisor. thank you very much.

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