You will always get a transcript, as the insertion does not stop transcription. It will probably stop translation, though. You need to sequence the full-length cDNA from your animals to ensure that you have the correct KO. And, you need to do a western blot. Since you have expression, you will have to confirm that you do not have protein to publish this. Also, if I were you, I'd figure out a way to genotype by Southern blot. Most labs that do KO insist that their KOs be confirmed by Southern, not PCR.

I thought of mass spectroscopy from tissue lysates but that would be too expensive and time consuming

Hi Theodore,

Have you already checked by western blot? My initial response would be to assess lysed tissue from the brain, spleen and lungs of the KO versus WT mice and use a neuronal cell line (or cell line with a similar characteristic to one of the tissues you know it's expressed in) with and without siRNA or shRNA to your gene as a control. By western you should see knockdown of your gene via siRNA/shRNA and you can compare that to the tissue results from the mice to check if the protein is being produced. If the cassette is indeed disrupting the gene, you may find the size of the protein changes.

Let me know if you've tried this already and, if you have, I'll have another think.

Best wishes

Emma

Did you use primers corresponding to a full-length transcript? You should sequence the mRNA you detect by qPCR and see what that will tell you..

Thank you for your valuable answers. The primers we used were not for the full length transcript. They amplified a part of the mRNA downstream of where we know the cassette was inserted (based on the company's datasheet and genotyping).

Why would full-length transcript sequencing be informative to us? It would only prove exactly where the cassette lies and what its sequence is.

At a protein level, we are waiting for a specific antibody for this receptor, but I m afraid this will not be much useful, either as it will bind to any protein only if it can reach its epitope. It is possible though that because of the cassette insertion to the gene, the ORF might have changed slightly and give false-negative results, although a functional protein may still be produced.

Let me guess. You don’t know the sequence of the transcript you detect by qPCR. You don’t know the copy number of your gene or whether or not it has a pseudogene somewhere in the genome. You don’t have a biochemical assay to measure the Kd which may give you an idea if it is the same receptor or it is a related one, provided they display different affinity for the ligand. You don’t have a specific test to detect the protein, etc…I may have forgot something else. A functional assay could be useless if the function of your receptor is redundant as you have correctly suggested.

I don’t know what method you did use for genotyping and what exactly it tells you.

I think it is important to know the sequence of the transcript that you are detecting and not rely on deductions. The mature transcript will tell you the identity of the protein that is produced. Normally, a full mature transcript could be devoid of the cassette.

You can learn a lot from the example of the KO of Dnmt1. For years the scientific community thought that we were dealing with a true KO. They deleted exons 2-5 and put out of frame the rest of the ORF. Somehow the cells managed to make a partial functional protein which allowed them to survive.

Everything you say above is correct; we came up with a huge problem and have to start from scratch and re-define the information course from the DNA to proein. I know the sequence of the RNA transcript I detected but not because I sequenced it; rather due to bioinformatics. There are biochemical assays to test the Kd but they are not specific for our receptor, so we would have false positive results.

Genotyping included one primer specific for the cassette and a primer pair specific for WT gene. As a result, if the cassette was inserted, I got a larger band than when the cassette was absent.

Your last remark is sth I agree with you. Im afraid that the cells make a functional, maybe shorter protein, and this is why my functional test was positive. Thanks for your input, much appreciated!

Hi Theodore,

You’re welcome! I find your thread very interesting and I will be following it to see how you do deal with it and also on inputs you will receive.

If you mice strain produces shorter version of your protein then you have very little choices. If you raise an antibody it will only tell you that: a shorter version. I don’t think MS identification is worth to try in this case.

If you sequence the mature transcript you will be able to deduce the amino acid sequence of the protein if it is produced and you will be able to understand what happened with the cassette because you have a map. You cannot rely to deduce the sequence of your transcript only from its genomic sequence and the bioinformatics. A lot of things might happens at the level of splicing, etc…It makes me remember the Jeff Goldblum expression in Jurassic park “Life finds a way”

Western blotting is the golden standard and nothing else... This is piece of money has to be spent...

If the qPCR primer you used is downstream of the cassette, it is very likely you'll see a signal, since the mRNA is indeed been made. Do you know whether the inserted cassette cause a frame shift (a common strategy in making KO mice) in the original gene? If that's so, no functional protein will be synthesized. Otherwise it'll be very difficult to prove that the truncated protein has no function.

Another question is which selective agonist did you use for your functional assay? Is it really that specific?

Hi Theodore,

Indeed there is a lot of missing information about the strategy used for generating the mice. The insertion site, how many exons are targeted as well as possible in frame splicing are things that you should consider when generating such animal models. Again genotypying strategie is also a critical point, if all your strategy relies on PCR you should be aware that "internal" primers pair should also give positive signal although the site has not being properly targeted ! Indeed if you PCR primers are in the construct, it could also match for non targeted loci (random insertion).

By western is indeed I pretty common way to verify couple of things (if you are not dealing with proteins that are not too redundant and the antibody have been shown specific for your target). If you have a frame-shift you could anticipate a slower migrating band, be careful of alternative splicing and promoter usage ! So basically, you could have several different things explaining your problem.

It would be good to know what the nature is of the so called "KO".

If this comes from a company, chances are high this is a gene trap cassette which is based on splicing. However, splicing is not necessarily 100%, so after skipping of the gene trap, WT protein may have been made.

You need to enquire from the company about their claim of sending you the right KO. An immunoblot will give you a good answer. You can also sequence your PCR products and find out.

Hi again, I agree with Koen and the problem of having a gene trap is that it could generate some splice around thus only making hypomorph mice (in the best case for you) without any deleterious effect ! Better use of gene trap are promoter or pA trapping stratregy

We bought the mice from a big US laboratory and they were generated by insertion of a cassette to the 191-525bp of the mRNA by homologous recombination. Based on the manufacturer's info, parts of both exons of the gene were deleted by insertion of the cassette (exon1 is 1-216 and exon2 is 217-1836).

We ll know more details about what they did, as soon as we sequence the mRNA produced.

@Xin Xie: the selective agonists we used are indeed specific for this receptor and thats why the functional assay initially worried us.

Is the ATG within exon 1? If I were you I would run a PCR on mRNA starting from a position before 191 and a finishing after 525 to see whether you readly amplify something or not. And by genomic PCR to see whether the intron 2 is really deleted but also the boundary between exon1/intron2 and intron2/exon2. If you know exactly where is the boundary between the targeting vector and the locus, I will also design a genomic PCR to see this boundary too... thus one primer inside the recombisaison arm and the second outside the targeting construct.

Hope this help

Daniel

You will always get a transcript, as the insertion does not stop transcription. It will probably stop translation, though. You need to sequence the full-length cDNA from your animals to ensure that you have the correct KO. And, you need to do a western blot. Since you have expression, you will have to confirm that you do not have protein to publish this. Also, if I were you, I'd figure out a way to genotype by Southern blot. Most labs that do KO insist that their KOs be confirmed by Southern, not PCR.

Pick up a copy of Manipulating the Mouse Embryo http://books.google.com/books/about/Manipulating_the_Mouse_Embryo.html?id=4juoa5xMs8oC. It should be mandatory reading for anyone using KO mice in their studies.

Amy,

Yes, I think it should be mandatory to check the KO in mice or whatsoever organism by southern blot, even if there is a low probability for non-homologous recombination to happen it can happen. It will also tell if it is homozygote, heterozygote, etc.... Just imagine an extra-integration of the cassette somewhere in the genome which could have profound phenotypic alterations that have nothing to do with our gene of interest.

all the comments above are very smart. one other thing you can consider is to align all the cannabinoid receptor protein sequences in Uniprot to determine whether your receptor has unique epitopes. You can then check for available antibodies specific to that epitope or buy one specifying that epitope. Having an antibody made may seem pricy but it shouldn't be nearly as much as generating a KO mouse! Similarly, IP-WB using existing Abs of the KO versus the WT and Het samples should also reveal differences at the protein level caused by the deletion. good luck!

Hi Theodore,

You mentioned that you ran a qPCR and discovered mRNA transcription in all tissues in which the receptor is expressed in WT mice; spleen, brain and lungs.

Did you use a Taqman probe based or SYBR green detection? I know that for each gene Taqman probes are available that target the different regions of the gene. You could confirm your results using probes that target other portions of the same gene.

As suggested by Daniel you could also run a PCR on mRNA starting from a position before 191 and finishing after 525 to check for amplification.

Hope you will resolve this.

You should still sequence you rqRT- PCR products at least once to double check. And, although there tends to be different TaqMan probes for many genes, if you look closely, you will see that most amplify similar regions. And, as mentioned before, this KO removes a few exons, so you will still see mRNA when doing qRT-PCR experiments.

If you can find an antibody for your cannabinoid receptor that works well in westerns, do it. Verify if the protein is expressed, if it is full-length or truncated, etc. The protein product is what really matters.

Regarding the response to treatment with specific agonist, just make sure that the agonist is pure and there is no contamination ( like LPS). To detect the expression of protein I agree with Paula for using a western in which instead of an antibody you can use the specific agonist attached to biotin or any chemiluminescent (if available).

Be sure that the agonists are truly selective at the concentration you are using. CB1 receptor agonists and antagonists in particular are known to have off target effects at high concentrations, such as cross-activity on GPCRs that signal through overlapping G protein pathways (e.g. Gi/o). In other words, the mutation may be correct as indicated by the genotyping assay, but the functional assay may be misleading. You should also try multiple agonists to validate the functional data.

FYI, if you are looking for cannabinoid receptor antibodies Dr. Kenneth Mackie is the world expert and many cannabinoid researchers around the world get them from him.