A practical approach to T-cell receptor cloning and expression.

Although cloning and expression of T-cell Receptors (TcRs) has been performed for almost two decades, these procedures are still challenging. For example, the use of T-cell clones that have undergone limited expansion as starting material to limit the loss of interesting TcRs, must be weighed agains...

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Autores principales:	Sébastien Wälchli, Geir Åge Løset, Shraddha Kumari, Jorunn Nergård Johansen, Weiwen Yang, Inger Sandlie, Johanna Olweus
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2011
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/79e97478b688499e9e0759a09d0b6d41
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spelling	oai:doaj.org-article:79e97478b688499e9e0759a09d0b6d412021-11-18T07:33:47ZA practical approach to T-cell receptor cloning and expression.1932-620310.1371/journal.pone.0027930https://doaj.org/article/79e97478b688499e9e0759a09d0b6d412011-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22132171/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Although cloning and expression of T-cell Receptors (TcRs) has been performed for almost two decades, these procedures are still challenging. For example, the use of T-cell clones that have undergone limited expansion as starting material to limit the loss of interesting TcRs, must be weighed against the introduction of mutations by excess PCR cycles. The recent interest in using specific TcRs for cancer immunotherapy has, however, increased the demand for practical and robust methods to rapidly clone and express TcRs. Two main technologies for TcR cloning have emerged; the use of a set of primers specifically annealing to all known TcR variable domains, and 5'-RACE amplification. We here present an improved 5'-RACE protocol that represents a fast and reliable way to identify a TcR from 10(5) cells only, making TcR cloning feasible without a priori knowledge of the variable domain sequence. We further present a detailed procedure for the subcloning of TcRα and β chains into an expression system. We show that a recombination-based cloning protocol facilitates simple and rapid transfer of the TcR transgene into different expression systems. The presented comprehensive method can be performed in any laboratory with standard equipment and with a limited amount of starting material. We finally exemplify the straightforwardness and reliability of our procedure by cloning and expressing several MART-1-specific TcRs and demonstrating their functionality.Sébastien WälchliGeir Åge LøsetShraddha KumariJorunn Nergård JohansenWeiwen YangInger SandlieJohanna OlweusPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 6, Iss 11, p e27930 (2011)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Sébastien Wälchli Geir Åge Løset Shraddha Kumari Jorunn Nergård Johansen Weiwen Yang Inger Sandlie Johanna Olweus A practical approach to T-cell receptor cloning and expression.
description	Although cloning and expression of T-cell Receptors (TcRs) has been performed for almost two decades, these procedures are still challenging. For example, the use of T-cell clones that have undergone limited expansion as starting material to limit the loss of interesting TcRs, must be weighed against the introduction of mutations by excess PCR cycles. The recent interest in using specific TcRs for cancer immunotherapy has, however, increased the demand for practical and robust methods to rapidly clone and express TcRs. Two main technologies for TcR cloning have emerged; the use of a set of primers specifically annealing to all known TcR variable domains, and 5'-RACE amplification. We here present an improved 5'-RACE protocol that represents a fast and reliable way to identify a TcR from 10(5) cells only, making TcR cloning feasible without a priori knowledge of the variable domain sequence. We further present a detailed procedure for the subcloning of TcRα and β chains into an expression system. We show that a recombination-based cloning protocol facilitates simple and rapid transfer of the TcR transgene into different expression systems. The presented comprehensive method can be performed in any laboratory with standard equipment and with a limited amount of starting material. We finally exemplify the straightforwardness and reliability of our procedure by cloning and expressing several MART-1-specific TcRs and demonstrating their functionality.
format	article
author	Sébastien Wälchli Geir Åge Løset Shraddha Kumari Jorunn Nergård Johansen Weiwen Yang Inger Sandlie Johanna Olweus
author_facet	Sébastien Wälchli Geir Åge Løset Shraddha Kumari Jorunn Nergård Johansen Weiwen Yang Inger Sandlie Johanna Olweus
author_sort	Sébastien Wälchli
title	A practical approach to T-cell receptor cloning and expression.
title_short	A practical approach to T-cell receptor cloning and expression.
title_full	A practical approach to T-cell receptor cloning and expression.
title_fullStr	A practical approach to T-cell receptor cloning and expression.
title_full_unstemmed	A practical approach to T-cell receptor cloning and expression.
title_sort	practical approach to t-cell receptor cloning and expression.
publisher	Public Library of Science (PLoS)
publishDate	2011
url	https://doaj.org/article/79e97478b688499e9e0759a09d0b6d41
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A practical approach to T-cell receptor cloning and expression.

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