Abstract
BackgroundAmplification and cloning of naïve T cell Receptor (TR) repertoires or antigen-specific TR is crucial to shape immune response and to develop immuno-based therapies. TR variable (V) regions are encoded by several genes that recombine during T cell development. The cloning of expressed genes as large diverse libraries from natural sources relies upon the availability of primers able to amplify as many V genes as possible.ResultsHere, we present a list of primers computationally designed on all functional TR V and J genes listed in the IMGT®, the ImMunoGeneTics information system®. The list consists of unambiguous or degenerate primers suitable to theoretically amplify and clone the entire TR repertoire. We show that it is possible to selectively amplify and clone expressed TR V genes in one single RT-PCR step and from as little as 1000 cells.ConclusionThis new primer set will facilitate the creation of more diverse TR libraries than has been possible using currently available primer sets.
Highlights
Amplification and cloning of naïve T cell Receptor (TR) repertoires or antigenspecific TR is crucial to shape immune response and to develop immuno-based therapies
We report a novel set of primers predicted to amplify nearly 100% of all functional TR V genes. We show that these primers can amplify transcribed TR V genes from as little as 1000 peripheral blood T cells, allowing a reliable and efficient method to clone TR repertoires
Data analysis and primers design The creation of large diverse libraries representing the specificities of TR repertoires relies on primers which are able to amplify all sequences coding for functional variable regions
Summary
Amplification and cloning of naïve T cell Receptor (TR) repertoires or antigenspecific TR is crucial to shape immune response and to develop immuno-based therapies. TR variable (V) regions are encoded by several genes that recombine during T cell development. The T cell receptor (TR) is a complex of trans-membrane dimeric proteins that mediate the antigen-dependent activation of T cells [1]. Most of circulating T cells express TR comprising of alpha and beta chains, while a minimal portion express the gamma and delta dimers [2]. Each chain consists in its extracellular region of a variable (V) and a constant (C) domain. Like immunoglobulin (IG), TR are encoded by several genes that undergo somatic recombination during T cell development [3]. According to the sequences deposited in IMGT®, the ImMunoGeneTics information system®, http:/ /imgt.cines.fr, [4,5,6], the human TRA locus has 47 TRAV, 50 TRAJ and 1 TRAC genes, whereas the TRB locus has 54 TRBV, 2 TRBD, 14 TRBJ and 2 TRBC genes; the TRD locus has 3 TRDV, 3 TRDD, 4 TRDJ and 1 TRDC genes, whereas the TRG locus has 9 TRGV, 5 TRGJ and 2 TRGC genes
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