I work on site-specific recombination, but on plants.

I think you can cross homologous line of tm1a/tm1a with homologous recombinase-expressing line FLPe/FLPe, so the F1 will have 1 copy of tm1a and 1 copy of FLPe in each cell. FLPe should work on tm1a and excise out the DNA fragment flanked by two FRT sites to produce tm1c structure. It depends on how efficient of recombinase FLPe, you might have a portion of cells with un-excised allele (tm1a). In this case, the F1 individual will be 'chimeric'. You will have to breed for further generation to obtain homogeneous 'excised-form' (tm1c) mouse line.

Thanks a lot! I did not consider the necessity of breeding one generation further but it makes complete sense. 

Hi Ariadne, to my view it is not necessary to use homozygous animals. Het animals should be enough, clearly offspring hast to be screened for the transgene. Mosaicism can get an issue (usually not with Cre, but with Flp I dont know), but if you find the Flp-recombined allele in the tail DNA usually there is a pretty good chance that the some of the offspring will also carry the recombined allele. These het recombined animals can be crossed to homozygosity, however, later on the tissue specific (?) Cre line will have to be crossed in, too. A long way to go! 

Right, there is no necessary to use homozygous animals. I just assumed that the mouse lines  Ariadne Penalva got for her experiments from other sources might have already been bred into homozygous status for those genes. If not using homozygous animals for both alleles, you will have to screen the F1-generation individuals with both alleles (tm1a and FLPe).

From your diagram, if your goal is to obtain mice with allele tm1d from tm1a, I am wondering--can we take an embryo (at 1-celled stage to avoid mosaicism) with allele tm1a, and inject both Cre and FLPe recombinase into it simultaneously to obtain tm1d structure, and culture these embryos into adult mice? Then, these mice should be homogenous for the tm1d allele. But, the worry is that the Cre might work more efficient and  faster than FLPe and remove one of the FRT sites first. Without this site, DNA fragment flanked by them cannot be excised by FLPe.

Thank you both for your throughout answers! Yuan-Yeu you are exactly right, my goal is to ultimately make a tm1d mouse from tm1a. Even though your suggestion is definitely very clever, I don't know if it will in the long run lead to more troubleshooting than crossing the mice? (Though it would take longer to get my desired transgenic if I do it through crossing generations...) 

Yes, I agree with you @Ariadne. As they say.....it is better 'Slowly but Surely'.

The method proposed could potentially involve some troubleshooting, such as (1) mosaicism (different stage embryos used), (2) toxicity of recombinase when both Cre and FLPe are expressed simultaneously, and (3) Whether one of the RFT sites  will be removed by Cre (mentioned earlier).......  However, we won't know these until experiments are done......

One good news is that I found one publication (Nature Genetics) saying that both FLPe and Cre are highly active enzymes in mice. Paper title: "High-efficiency deleter mice show that FLPe is an alternative to Cre-loxP"  [ http://www.nature.com/ng/journal/v25/n2/full/ng0600_139.html ]

By the way, this is just for your info (or future reference): nowadays, to create a conditional knockout mouse cells seem much easier through the assistance of genome-editing tools (such as CRISPR/Cas9 and TALEN).

Attached is a paper describing such method. The authors used TALEN to drop in two loxP sites flanking an exon of a gene to create conditional knockout mouse embryonic stem cells. They further simplified the experiment procedure by dropping the two loxP sites using synthesized loxP-ssODN (116 bp).  ssODN: single-stranded oligodeoxynucleotides.

And, theoretically, you can also drop in a loxP-ssODN at a precisely location outside the left FRT site of tm1a, and remove anything in-between through Cre to create tm1d mouse.

Paper title: Single-step generation of conditional knockout mouse embryonic stem cells https://www.ncbi.nlm.nih.gov/pubmed/26190102

Hi Ariadne Penalva,

Sorry for the late reply. I saw you posted a question for me earlier but just deleted today. But I still remember the question.

We verify the final product by checking whether they are  homogenous for the excised allele (in your case, tm1d) by using PCR, and then Southern. We use PCR to check excision junctions. Please see the attached PPT for an example. In the PPT, you can see the possible locations of 4 PCR primers (P1, P2, P3 and P4). You can use P1+P2 to amplify the final product ('tm1d'). If excised, a short and predicted PCR product should be amplified. Of course, you have to sequence the PCR product to make sure they match your predicted sequence. Afterwards, you want to check whether your mice have 'chimeric cells' (contain both non-excised allele and excised allele). You can use P1+ P3 and P2 + P4 for PCR amplification. If chimeric cells present, PCR bands from P1+ P3 and P2 + P4 will be amplified. Finally, we use Southern to re-confirm the PCR-correct lines to see whether the DNA fragment flanked by recombination sites (in your case, FRT/loxP sites) have been removed.

Sorry, I did not realize I deleted the question. Thank you very much, this will be very useful! :) 

Also, is the genetic background of the (FLPe and Cre) recombinase-expressing mouse line ('X') the same as that of tm1a-carrying mouse line ('Y')? I guess if they are different, then after you cross X and Y to obtain tm1c-carrying mouse line, and further cross to obtain tm1d allele-carrying mouse line, you have to do some backcross breeding to obtain a tm1d mouse with 'Y' mouse genetic background. Only under the same genetic background, can the comparison of the gene (tm1a) and its truncated form (tm1d) make meaningful, am I correct?