Hi Changun Yun,

this question is not easy to answer as I am not sure that I have all the information. An important information would be if your gene of intereset has only two exons or are there more exons?

However, it sound like parts of you cd lay in Exon 1 - this means if you knock out Exon 2 you still have left the transcription initiation site and the translation initiation site intact and transcription and even translation initiation might take place (depending on the stability of the mutated transkript).

However, if there are only two exons and you removed exon 2 you could get only a 7 bp peptide and I don't think you really would see this on the Western blot and most likely it has no function at all.

If there are more Exons behind Exon 2 there is at least the possibilty that there is a protein without Exon 2 which

1. can be unfunctional/lost function

2. can be partially functional

3. can still be functional

However, in this case you still need to have an ORF after removal of of Exon 2 (please check).

If something like this has happpend I would expect that the normal protein and the protein missing Exon 2 have different size, which you can hopefully see on the Western blot (there are programs out there calculating the size of a protein by its sequence).

What I also don't know - is there the possibility of different Protein Isoforms (this might also be only a problem if you have more that 2 Exons) due to differential splicing or differential transcription initiation?

Because, then there is the possibilty that you have isoforms which don't need Exon 2 at all ( I worked with a gene encoding two transcripts and you could remove two Exons from the gene and only affect one transcript - the other remained complety intact, leading to only a partial phenotype).

What I would recommed if you have more than 2 Exons:

1. check if there is an ORF after removal of Exon 2 (and the size of the protein you might get)

2. check if there are isoforms of the transcript and the protein (which might not need Exon 2)

If you have only the 2 described Exon and the antibody is working it seems that something is really off with the ko mouse. However, it also seems that you are not totally sure about the antibody you use for detection - this is a difficult situation in itself.

I think you have determined the ko on genome level - is this correct?

However - have you also checked on transcript level to see if the transcript is really absent or truncated ?

That can be easily done by isolation RNA, reverse transcription followed by PCR against your gene of interest (you should always include a positive control like actin or GAPDH). You could alsoo due Northen Blot.

If the knock out worked as it should and you have only 2 Exons there are two options

1. you detect no transcript at all (due to transcript instability)

2. You detect only a truncated transcript

Looking for the transcript might also help if there are more than 2 Exons because than you can design your primer so that you can distinguish between an amplicon contain Exon2 and an amplicon without Exon 2.

I hope this helps a little bit - if you have additional questions feel free to ask

First of all, thank you for your detailed answer.

The gene I want to KO has only 2 exons. After removing exon 2, the remaining CDS sequence of exon1 is only 21 bp (=7 amino acids). The intron of exon 1 also remains.

Since there are only 2 exons, I don't think differential splicing or differential transcription initiation will be a problem, as you answered.

I extracted genomic DNA and confirmed KO at the genome level through PCR, and I also confirmed that it was not expressed at all through PCR after RNA isolation.

And I think there will be no additional ORF, but there is an exon of a completely different protein behind the removed exon2 (although it is a little far away). I think it is unlikely, but is it possible that the remaining transcription initiation site or translation initiation site and the short CDS of exon1 can cause protein synthesis and detection by antibody?

Hi Changun Yun,

You have only these two exons and you should get only a 7 AS long peptide. Even if this peptide is dedected by (the unlikely) chance by your antibody I am not sure if you would see this on the Western Blot at this peptide should run really low in the blot (and should be clearly distinguishable from your wildtype protein by size).

In addition there is the chance that the removal of the second exon destabilizes the remaining mRNA so that this might lead to lesser or no translation at all.

How sure are you that the antibody you have is detecting the right protein?

Because in the moment for me it sounds more likely that you have an antibody problem here. Are they any alternative antibodies you can try? I would recommed to try this first because it is always possible that antibodies on Western blots are not 100% specific and you also mentioned that "The antibody used for WB is an antibody that is not certain to capture mouse proteins".

If not - can you test the specifity of your antibody by overexpressing your gene of interest (with a tag) and try to detected the overexpressed protein with your antibody (however this would be a lot of work)?

Another question - as you have hopefully a full knock out for your gene, did you see any phenotype in your knock out mouse which might distiguish you ko mouse from wt mouse? Phenotypes can be subtle and can affect anatomy, behavior, vitality, reaction to infection and more and are sometimes not easy to spot. If you find something here, this could indicate that you have indeed a ko mouse and that the antibody is the problem.

Something I have see once was a wrong gene annotation were two annotated genes were indeed one functional gene. But this was not in mouse and the mouse genome is very good characterized - so I don't think this will apply here (I am just mentioning ist because you mentioned the exon of another gene in proximity of your second exon).

KR Dörthe

Hi, Dörthe Andrea Kesper.

Actually, I'm not sure the antibody detects the right protein.

As you said, I confirmed the antibody's ability to detect the target protein using an overexpression experiment. Transfect the target gene overexpressing vector into 293T and the empty vector transfected 293T protein lysate.

The empty vector transfected 293T sample didn't detect the target protein, and the target gene overexpressing vector transfected 293T sample detected the target protein.

But, as I said in my first question, primary cells from WT or KO mice showed weak bands even in the absence of inflammatory stimuli.

(I've attached a photo.)

Also, I tested the antibody that was produced by another company, but never detected that target protein.

And I know that the KO mouse has no phenotype that can be distinguished from the WT mouse.

Thank you

Hi, Changun Yun. In addition to what Dörthe Andrea wrote, I would consider the possibility of the gene having some other copy in the genome of the mouse. A southern blot would be able to tell if that's the case. I don't know if a 7 amino acids peptide could fold in a way that allows detection by the antibody. Do you know where the antibody binds normally? You may also want to try a KO eliminating both exons. Good luck with your experiments.

Hi Changun,

from your overexpression experiment ist sounds that the antibody is recognizing your protein - however it looks like your knock out protein runs at the same hight as the wild type protein and I am quite sure that this should not happen when you have removed large part of the protein. You can use "Protein Molecular Weight" to calculate the size of your wild type proteine versus the knock out protein.

Do you know again which part of the protein your antibody was raised? You should check this. If the antibody was not raised against the first 7 AS of your protein of interest it is quite unlikely that the antibody would dedect your truncated protein.

As Francisco Javier Alvarez it could be that there is another copy of the gene or a very similar in the gene in the genome. Did you blast you gene against the mouse genome and searched for similar genes which could be redundant?

Are you willing to share information about the gene you have mutated? Than it might be that I can give you some more information (but I understand when you don't want to share this - and I can give you no guarantee that I can solve the problem).

If you want to do the knock out again make sure to delet the translational start site and if you know the transcriptional start site it might be good to delet this site as wel.

KR Dörthe

Francisco Javier Alvarez I have verified that the gene I KO has no other analogues. The antibody targets the full length of the target protein. I will try antibody testing on several different cell lines. Thank you for your advice.

Dörthe Andrea Kesper Hello.

First of all, the photo I attached is not overexpressed, but the target protein at the endogenous level was confirmed in the primary cell.

It is said that the antibody I used was produced after binding GST to the full-length target protein.

Also, I checked through NCBI and ensemble genome browser to see if there were other copies of the gene I was trying to KO, but I confirmed that there were none.

I will try to test the antibody on another mouse cell line.

Thank you for your continued help and advice.