Abstract
Transgenic expression of B- and T-cell receptors (BCRs and TCRs, respectively) has been a standard tool to study lymphocyte development and function in vivo. The generation of transgenic mice is time-consuming and, therefore, a faster method to study the biology of defined lymphocyte receptors in vivo would be highly welcome. Using 2A peptide-linked multicistronic retroviral vectors to transduce stem cells, TCRs can be expressed rapidly in mice of any background. We aimed at adopting this retrogenic technology to the in vivo expression of BCRs. Using a well characterised BCR specific for hen egg lysozyme (HEL), we achieved surface expression of the retrogenically encoded BCR in a Rag-deficient pro B-cell line in vitro. In vivo, retrogenic BCRs were detectable only intracellularly but not on the surface of B cells from wild type or Rag2-deficient mice. This data, together with the fact that no BCR retrogenic mouse model has been published in the 7 years since the method was originally published for TCRs, strongly suggests that achieving BCR-expression in vivo with retrogenic technology is highly challenging if not impossible.
Highlights
Over the last three decades transgenic mice have been valuable tools to study the biology of lymphocytes
We show for the first time the expression of a recombinant, membrane IgM-B-cell receptor (BCR) in vitro using the pro-B cell line R5B, which is deficient for endogenous Ig chains
Cloning of the recombinant hen egg lysozyme (HEL)-IgM B-cell receptor To establish the generation of BCR retrogenic mice we decided to use the Hen-Egg-Lysozyme (HEL)-specific membrane form of m heavy chain (Igm) and the kL chain (Igk) of the BCR MD4
Summary
Over the last three decades transgenic mice have been valuable tools to study the biology of lymphocytes. Breeding transgenic mice onto different backgrounds either by conventional back-crossing or the speed congenic approach is time consuming and expensive To overcome these major limitations, a new technique to express TCRa and TCRb chains from a 2A peptide-linked bicistronic retroviral vector using retroviral-mediated stem cell gene transfer was developed and published in 2006 [4,5,6]. Retrogenic mice cannot be propagated by breeding, because there is no germline transduction and the analysis is limited to the hematopoietic system [4] Another major advantage of the retrogenic approach is the usage of so-called 2A peptides to link two or more target proteins instead of an IRES. We detected the recombinant aHEL IgM-BCR intracellularly when analysing these retrogenic mice, but to our surprise, we failed to demonstrate the surface expression of the recombinant aHEL IgM-BCR in vivo
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