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Mouse Germline Hybridoma Sequences?


Best Answer pdbuab, 15 April 2015 - 10:04 AM

Hi,

The most complete info about Ig genes is IMGT http://www.imgt.org/

An easy way to search it though is using NCBI Ig Bast http://www.ncbi.nlm.nih.gov/igblast/

The closest hit to sequence 1 is:                
V  77.8% (70/90)  IGKV4-59*01 This looks to be a highly somatically mutated Vk
Sequence 2:

V  98.9% (87/88)  IGKV6-13*01 There is only aa difference L14I between your sequence and the germline sequence. This could be a polymorphism.

If you are only getting one VH sequence it is pretty unlikely that this hybridoma is biclonal. It is also unlikely that the transcript is coming from an unrearranged germline Vk gene segment since 1) Such "sterile" transcripts are not usually found in plasma cells and would be at very low levels if present, and 2) You have quite a bit of sequence C' of the end of the germline gene sequence (it ends SPPP in sequence 1 and SSYP in sequence 2.) This is likely Jk sequence and thus both sequences are coming from VJk rearranged alleles. Is your downstream primer in Ck?

It is possible that one of these transcripts is coming from a non-productively rearranged allele, although usually those transcripts are unstable due to nonsense mediated mRNA decay.  Are you sure there is no stop codon in sequence 2?

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#1 SD2433

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Posted 15 April 2015 - 04:54 AM

Hi everyone,

 

New user here, and I am hoping to get some insight on a research problem we have been having in my lab.  I have been working on determining the sequences of antibodies expressed in mouse hybridoma cell lines using a variety of primer sets targeting the heavy chain variable and light chain variable (Vk) regions.  When sequencing the Vk region of one particular hybridoma, I consistently get 2 distinct sequences with my primer panel, despite doing limited dilutions and attempting to get a monoclonal population.  This makes me think that this strain is polyclonal despite our efforts at doing limited dilutions, unless the sequences belong to the mouse germline.  I've tried checking these sequences against known mouse germline and mouse pseudogene sequences but am not getting any matches.  Has anyone here seen these sequences come up in their work as known mouse germline genes?  If not, do you know of any good databases where I could try to match these sequences to the mouse germline?

 

Thanks in advance for your help!  The amino acid sequences are below

 

Sequence 1:

QSPAIISAFTGEKVTMTCSARSSESCMHWLQQRSGTFPKRWINDTSKPTSGVPVRFSGSGSGTSYCLSISSVESVDAATYYCQQWRSPPPRGRSVEAPSWKSNGLMLH

 

Sequence 2:

QKFMSTSVGDRVSLTCKASQNVGTAVAWYQQKPGQSPKLLIYSASNRYTGVPDRFTGSGSGTDFTLTISNMQSEDLADYFCQQYSSYPLTFGAGTKLELRRADA



#2 pdbuab

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Posted 15 April 2015 - 10:04 AM   Best Answer

Hi,

The most complete info about Ig genes is IMGT http://www.imgt.org/

An easy way to search it though is using NCBI Ig Bast http://www.ncbi.nlm.nih.gov/igblast/

The closest hit to sequence 1 is:                
V  77.8% (70/90)  IGKV4-59*01 This looks to be a highly somatically mutated Vk
Sequence 2:

V  98.9% (87/88)  IGKV6-13*01 There is only aa difference L14I between your sequence and the germline sequence. This could be a polymorphism.

If you are only getting one VH sequence it is pretty unlikely that this hybridoma is biclonal. It is also unlikely that the transcript is coming from an unrearranged germline Vk gene segment since 1) Such "sterile" transcripts are not usually found in plasma cells and would be at very low levels if present, and 2) You have quite a bit of sequence C' of the end of the germline gene sequence (it ends SPPP in sequence 1 and SSYP in sequence 2.) This is likely Jk sequence and thus both sequences are coming from VJk rearranged alleles. Is your downstream primer in Ck?

It is possible that one of these transcripts is coming from a non-productively rearranged allele, although usually those transcripts are unstable due to nonsense mediated mRNA decay.  Are you sure there is no stop codon in sequence 2?



#3 SD2433

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Posted 20 April 2015 - 12:17 PM

pdbuab,

 

Thank you very much for taking the time to reply!  I am new to the antibody world and was not aware of NCBI Ig Blast; this will be a very useful tool going forward. 

 

In light of this information, it certainly does seem that Seq2 is likely a germline gene.  To answer your questions, our downstream primer is in the Ck region.  We are not sure whether a stop codon is present in sequence 2, but thanks for raising that point, that is a possibility and eventually we may do some mass spec-based protein sequencing if necessary.  Another problem we have been encountering with this hybridoma, and that contributed to our confusion, is that we have been unable to get any VH sequences, despite using multiple primer sets that have otherwise been reliable.  So there is still work to be done here.

 

Thank you again for your very helpful reply!






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