Hi Yuka,

To fully KO your GOI, you'll need your experimental animals to be homozygous for the floxed allele. One copy of the cre driver allele is usually enough, on the other hand.

So, IMO, you'd want to mate your F1 generation (all double hets) with the homozygous floxed parent. If all goes well, you'll be able to pull out your ideal breeders (homo floxed male x homo floxed homo cre females) from that.

Once you have that setup, you can simply use all the [fl/fl, CRE/+] offspring without even needing to genotype!

To easily renew your breeders (or in case you need wt controls that are littermates of your cKO mice) you can mate the experimental animals together, offspring you'll get will be all fl/fl, and:

25% CRE/CRE (to renew female breeders),

50% CRE/+ (for expts),

25% +/+ (for wt control expts or to renew male breeders).

Hope this helps!

Best,

Alberto

Hi Albert, thanks a lot for your advice!

Unfortunately, mendelian genetics doesn't really work on Camkiia-Cre transgenic model. I can barely get Cre+;fxfx mice (both male and female).

I've actually done up to F2 generation, yet there is no male Cre+;fxfx and only 2 out 92 mice are Cre+;fxfx female.

As you suggested, I crossed F1 Cre+;FxWT with fxfx for both male and female cre drivers. Since I can barely get homozygous floxed mice, I crossed hets for F2.

Anyway, this is why I would love to hear suggestible breeding scheme, specific to Camkiia-Cre transgenic mice model.

Hi again Yuka,

there is no special scheme for CaMKIIa-Cre mice. First of all, there are several different lines for this Cre-driver, I'm aware of 2 that are transgenic and one knock-in. Depending which one you have, the behavior of the Cre allele is different. In any case, unfortunately the interaction of the Cre allele with the specific locus you need to delete is every time different. So, for example we have loci that are deleted in germline when our CaMKII-Cre allele is in females and other loci that are deleted if the same Cre allele is in males. This is something you will have to determine in your specific case if it happens and in which gender.

In general your mating strategy is fine, once you are sure you have no germline deletion you will cross a flx/flx;Cre/+ animal with a flx/flx animal and obtain theoretically 50% of each as offspring.

On the other hand, there are genetic reasons why you might obtain non-Mendelian ratios of mice from the F1 matings (fx/wt;Cre/+ with flx/flx mice):

1. Your allele is highly deleted in the germline of the animal carrying the Cre, meaning that you will obtain del/flx or del/wt animals, with or without the Cre: if you don't check for the deleted allele you might think that you obtain flx/flx or wt animals, but they are not... But I don't think this applies to you, because you don't see any, or very few flx/flx animals with the Cre, but many flx/wt!

2. The Cre allele is on the same chromosome as your GOI... In these cases you have to count on crossing over to co-segregate the Cre locus and the GOI, because you can put them together easily in heterozygosity with one allele containing the floxed gene and the other allele the Cre, but to have a flx/flx;Cre/+ mouse, you need to bring on the same allele the two loci, and this can only occur by crossing-over between the two alleles during meiosis: the closer the Cre and the GOI are, the less likely they will cross-over! But if you manage to get them together, then they will likely also remain together, co-segregating in most of the F2 offspring! So, if the 2 females you say are flx/flx;Cre/+ have been checked for the del allele and they don't have it in the tail tip or ear-punch (whatever you use for genotyping... just to make sure, they might be actually del/flx, which is still fine, since the complete ko you would only have in CaMKII-Cre expressing cells, other cells will be het, but that's normally tolerated), I would definitely mate these with a flx/flx male and if you obtain Mendelian ratios of the same parental genotypes in F3, you'll know this is your case... rather unfortunate, I must say, but it could have been worse, if you hadn't obtained any flx/flx;Cre/+ at all!

3. I don't know what gene you are working on, and it is probably a rare case, but the deletion of your gene in the anterior brain might be lethal to the mice... You can still find escapees or survivors, for whatever genetic compensation mechanism, of course, but they would be very very few, if any. Normally CaMKII starts being expressed at P19, but some transgenic drivers start even earlier, so you should notice death occurring around this time, which I don't think I understand is your case... But just to keep in mind, in general.

Again good luck!

Pietro Pilo Boyl Thank you very much for detailed explanation! I also noticed that there are several Cakiia-cre mice are available. We purchased B6.Cg-Tg(Camk2a-cre)T29-1Stl/J one. There should be a couple of different ways to assess germ line deletion. Which methodology have you used for your Cre model?

Also, what do you mean by del/flx? Sorry for many questions by the way.

With respect to your third explanation, I count number of pups at P0 and at P19 (at weaning point). So far I don't see any deleterious effect of Cre-recombination during the early postnatal period. However, I noticed the litter size is very variable. Some of the mating pairs produce over 10 pups while others produce less than 5. I still haven't figured out the reasoning of this variation as the parents' genotypes are mostly Cre+;fx/wt.

Hi Yuka, I see, you use the Tonegawa mouse that is sold as mostly restricted to the CA1 region (we don't use this mouse line, we have one from Minichiello/Klein... But looking at the original paper - Tsien et al., Cell, 1996 -, which you surely have, it is not optimal the way they analysed the expression pattern of the line: check this link for a new expression analysis from 2011, probably by The Jackson Lab:

https://ndp.jax.org/NDPServe.dll?ViewItem?ItemID=2125&XPos=-13104910&YPos=2654494&ZPos=0&Lens=0.5&SignIn=Sign%20in%20as%20Guest).

So, your two questions:

1. How to assess germline deletion. Not difficult. I imagine you have a PCR to check deletion of your gene in the CA1, with sense primer upstream of the 5' LoxP site and anti-sense primer downstream of the 3' LoxP site (to amplify a band if the recombination has occurred): that's the del band. Try this PCR on your genotyping material (tail tip, ear punch....whatever you use). If you obtain the del band it means you have deleted earlier in development and not specifically. If the del band is present in the absence of the Cre, then you really have deleted in germline... in your case this would be particularly true, because the Cre and your floxed locus are probably close to each other on the same chromosome, but in the double hets highly likely on different alleles (you are having trouble segregating them together), so when you delete in germline the two alleles separate at latest during the last meiosis and you would get a germ cell with the deleted allele and no Cre allele. The germ cell will then be fertilized by one coming from the other parent, which would contain the flox allele (according to your present mating scheme, since it will be a homozygous flox mouse) and you obtain a mouse with genotype del/flx and no Cre. In your case it might not be terribly important, but you would need to look for offspring that co-segregates the Cre in order to use the animals for experiments. If you manage that, then you will have a heterozygous mouse (which will probably be viable and without major phenotype) that will become ko in the CA1 when the Cre is regularly reactivated. You should anyway use as controls in your experiments the del/flx mice without Cre.

2. Litter size is variable. This is a bit complicated and very specific, but you should not worry too much about it, just try and use littermates as control mice in your experiments. In practice it comes from the fact that the original CamKIIa-Cre line donated to The Jackson Lab by Tonegawa was not a pure background, but a mixture between C57Bl/6N and C57Bl/6J substrains. The first (among other differences, that's why I suggest littermates as controls) is more fertile than the second, so in the population, depending which traits are transferred to the female, you might get a more or less fertile one. Of course this is then transferred onto your double hets... At some point it should dilute out if your mice are in a clean background, but it will take some generations.

Cheers.

PS. Asking questions is a very good sign... is a sign of interest in what you are doing, is a sign that you observe and is a sign that you wish to understand what you observe. It never harms, it's always worth... At worst you might not get an answer, or a satisfying answer... But scientists should always ask questions, if anything to oneselves...