Methods and materials for cloning functional t cell receptors from single t cells

Abstract

This document provides methods and materials involved in cloning functional TCRs from single T cells. For example, methods and materials for obtaining nucleic acid encoding a TCR from a single T cell and arranging that nucleic acid to form nucleic acid vectors successfully designed to express a TCR, kits for obtaining nucleic acid encoding a TCR from a single T cell and arranging that nucleic acid to form nucleic acid vectors successfully designed to express a TCR, methods for making such kits, collections of nucleic acid primers designed to amplify the entire coding sequence of both variable regions for each expressed V segment for functional αβ or γδ TCRs of a particular mammalian species, methods for using such collections of nucleic acid primers to clone functional TCRs from single T cells, and kits containing such collections of nucleic acid primers to clone functional TCRs from single T cells are provided.

1 . (canceled) 
     
     
         2 . A method for obtaining a plurality of functional T cell receptors, the method comprising:
 (a) amplifying a first amplification product and a second amplification product from cDNA generated from RNA obtained from a single T cell sorted into each of a plurality of separate locations of a device, wherein said first amplification product comprises nucleic acid encoding an α variable (Vα) or γ variable (Vγ) segment, and said second amplification product comprises nucleic acid encoding an β variable (Vβ) or δ variable (Vδ) segment, and   (b) assembling said first amplification product and said second amplification product from each of said plurality of separate locations to obtain an assembled nucleic acid encoding a functional T cell receptor for each of said plurality of separate locations,   wherein said functional T cell receptor comprises (i) a full-length α variable region and a full-length β variable region from said single T cell or (ii) a full-length γ variable region and a full-length δ variable region from said single T cell.   
     
     
         3 . The method of  claim 2 , wherein the assembled nucleic acid encoding a functional T cell receptor is assembled into a nucleic acid vector. 
     
     
         4 . The method of  claim 3 , wherein said nucleic acid vector is a nucleic acid expression vector. 
     
     
         5 . The method of  claim 2 , wherein said plurality is greater than 50. 
     
     
         6 . The method of  claim 2 , wherein said device comprises a multi-well plate. 
     
     
         7 . The method of  claim 2 , wherein said single T cell is a single human T cell. 
     
     
         8 . The method of  claim 2 , wherein said first amplification product comprises nucleic acid encoding a leader (L) sequence of a Vα or Vγ segment. 
     
     
         9 . The method of  claim 2 , wherein said first amplification product comprises nucleic acid encoding a Jα or Jγ segment. 
     
     
         10 . The method of  claim 2 , wherein said first amplification product comprises nucleic acid encoding a 5′ portion of a Cα or Cγ region. 
     
     
         11 . The method of  claim 2 , wherein said first amplification product comprises nucleic acid encoding a leader (L) sequence of a Vα or Vγ segment, a Jα or Jγ segment, and a 5′ portion of a Cα or Cγ region. 
     
     
         12 . The method of  claim 2 , wherein said second amplification product comprises nucleic acid encoding a leader (L) sequence of a Vβ or Vδ segment. 
     
     
         13 . The method of  claim 2 , wherein said second amplification product comprises nucleic acid encoding a Dβ or Dδ segment. 
     
     
         14 . The method of  claim 2 , wherein said second amplification product comprises nucleic acid encoding a Jβ or Jδ segment. 
     
     
         15 . The method of  claim 2 , wherein said second amplification product comprises nucleic acid encoding a 5′ portion of a Cβ or Cδ region. 
     
     
         16 . The method of  claim 2 , wherein said second amplification product comprises nucleic acid encoding a leader (L) sequence of a Vβ or Vδ segment, a Dβ or Dδ segment, a Jβ or JE segment, and a 5′ portion of a Cβ or Cδ region. 
     
     
         17 . The method of  claim 2 , wherein said first amplification product comprises an adapter sequence added to an amplified template sequence of said cDNA via a second round amplification of said amplifying. 
     
     
         18 . The method of  claim 2 , wherein said second amplification product comprises an adapter sequence added to an amplified template sequence of said cDNA via a second round amplification of said amplifying. 
     
     
         19 . The method of  claim 2 , wherein said first amplification product comprises a first adapter sequence added to an amplified template sequence of said cDNA via a second round amplification of said amplifying, and wherein said second amplification product comprises a second adapter sequence added to an amplified template sequence of said cDNA via a second round amplification of said amplifying, wherein said first and second adapter sequence are different. 
     
     
         20 . The method of  claim 2 , wherein said functional T cell receptor of each of said assembled nucleic acid comprises a Vα/Vβ combination or a Vγ/Vδ combination from said single T cell. 
     
     
         21 . The method of  claim 2 , wherein said functional T cell receptor of each of said assembled nucleic acid comprises (a) a full-length α variable region and a full-length β variable region or (b) a full-length γ variable region and a full-length δ variable region. 
     
     
         22 . The method of  claim 2 , wherein said functional T cell receptor of each of said assembled nucleic acid comprises (a) a full-length α variable region and a full-length β variable region from said single T cell or (b) a full-length γ variable region and a full-length δ variable region from said single T cell. 
     
     
         23 . The method of  claim 2 , wherein said functional T cell receptor of each of said assembled nucleic acid comprises (a) a full-length α constant region and a full-length β constant region or (b) a full-length γ constant region and a full-length δ constant region. 
     
     
         24 . The method of  claim 2 , wherein said assembled nucleic acid comprises a nucleic acid sequence encoding a self-cleaving peptide or an internal ribosome entry site (IRES). 
     
     
         25 . The method of  claim 2 , wherein the method comprises sorting said single T cells into said separate locations. 
     
     
         26 . The method of  claim 2 , wherein the method comprises performing a reverse transcription reaction to obtain said cDNA. 
     
     
         27 . The method of  claim 2 , wherein seamless cloning is used for said assembling. 
     
     
         28 . The method of  claim 2 , wherein said assembled nucleic acid is obtained without performing nucleic acid sequencing. 
     
     
         29 . The method of  claim 2 , wherein said assembled nucleic acid is obtained without performing a restriction endonuclease cleavage reaction.