scFv cloning and expression - problem with pseudogenes

RVDESHP0 at pwinet.upj.com RVDESHP0 at pwinet.upj.com
Tue Apr 9 14:19:31 EST 1996


We went through all the problems that you have mentioned and MORE !  First 
of all, the pseudogene that you talk about produces abundant amounts of an 
aberrant light chain transcript that is NOT translated (because of the stop 
codon at the junction of CDR3/J region).  This problem originates from the 
myeloma cell fusion partner(s) used in generating monoclonal hybridomas.  
The two specific references describing exactly this phenomenon are :

1.      Hybridoma fusion cell lines contain an aberrant light kappa 
transcript.  W.L.Carroll et al, Molecular Immunology, 25:991-995, 1988.

2.      Immunoglobulin transcripts and molecular history of a hybridoma that 
produces antibody to carcinoembryonic antigen.  S. Cabilly and A. D. Riggs, 
Gene, 40:157-161, 1985.

The two other alternatives that we found to getting around this problem may 
be in these two papers :

1.      An improved method for generating single-chain antibodies from 
hybridomas.  P.J.Nicholls et al, J. Immunol. Meth., 165:81-91, 1993.

2.      An extended primer set for PCR amplification of murine kappa 
variable regions.  S.Leung et al, Biotechniques, 15:286, 1993.

We are currently working on the second option.  We haven't tried option 1 
yet.  I hope, this helps.

************************************************************************ 
Rajendra V. Deshpande, Ph.D.
Pharmacia & Upjohn, Inc.
Molecular Biology Research
Mail Stop 7242-209-724
301 Henrietta Street
Kalamazoo, MI 49007
Tel. (616)833-1755
Fax (616)833-1559
e-mail : rvdeshp0 at pwinet.upj.com
************************************************************************



______________________________ Reply Separator _________________________________
Subject: scFv cloning and expression - problem with pseudogenes
Author:  regina at biotech.ufl.edu (Regina Shaw) at INTERNET
Date:    4/9/96 3:33 PM


Hello,

We have isolated the light and heavy chains of the immunoglobulin genes 
for three hybridomas, for cloning and later expression. Our problem is 
that sequencing data show that all of them consistently have a frameshift 
at the junction of the CDR3 - J (1 base missing) so for some reason we are 
amplifying preferentially the pseudogenes only instead of the functional, 
in-frame genes. Initially we thought this was due to PCR errors (Taq) so 
we switched to Vent. Next we thought that using the same upper primer, but 
a different lower primer would help, so we did nested PCR (amplified the 
approx. 700 bp PCR fragment spanning the stretch between the 5' end of 
each light/heavy chain -> constant region), all with the same result.  - 
The hybridomas were originally produced by using spleen cells from BALB/c 
immunized mice, and PAI cells, and all three of them produce strongly 
functional antibodies (in immunofluorescence and on Western blots). - 
Total RNA was isolated using the Qiagen kit; for reverse transcription we 
used Invitrogen cDNA cycle kit, and then usually 30-40 rounds of (nested) 
PCR with Taq or Vent, using the primers adapted from Huse et al.
Would anybody have an explanation why we can't seem to amplify out the 
functional genes? Is it something to do with the PAI cell line maybe? 
Any comments and suggestions are greatly appreciated.

Thank you very much in advance for your help,

Regina Shaw
Recombinant Protein Expression Core/ICBR



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