RESUMO
The CD3epsilon proline-rich sequence (PRS) binds to the cytosolic adaptor molecule Nck after TCR ligation. It has been proposed that this interaction is essential for immunological synapse formation and T cell activation. To assess the physiological importance of the CD3epsilon PRS, we have generated mice that lack this motif (CD3epsilon.PRS(M)). Pull-down experiments demonstrated the inability of Nck to bind to the CD3epsilon PRS in thymocytes from mutant mice after TCR ligation. Surprisingly, no differences were observed in the number and percentage of T cell subsets in the thymus and spleen, and there was no apparent defect in positive or negative selection. Furthermore, the proliferative response of CD3epsilon.PRS(M) T cells to staphylococcal enterotoxin B and anti-CD3 Ab was normal. TCR surface expression, constitutive internalization, and Ag-induced down-modulation were also normal. These data suggest that the interaction between the CD3epsilon PRS and Nck, or any other Src homology 3 domain-containing molecule, is not essential for T cell development and function.
Assuntos
Complexo CD3/química , Proteínas Oncogênicas/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Sítios de Ligação , Complexo CD3/genética , Complexo CD3/metabolismo , Linhagem Celular , Proliferação de Células , Humanos , Técnicas In Vitro , Ativação Linfocitária , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Prolina/química , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/citologiaRESUMO
As science progresses in its understanding of diseases and their treatment, advances have been made in the biotechnology used in disease therapy. Most gene therapy approaches utilise viral vectors to deliver genes of interest. However, multiple proteins are often involved in disease processes and there is often a need to efficiently deliver more than one gene. Researchers have employed several strategies to accomplish this goal. When designing vectors to express multiple genes, there are several factors that need to be taken into account, including cell type, the activity of the protein of interest and subcellular protein localisation. In most cases, it is ideal for each protein to be expressed at comparable levels, a leading issue with traditional strategies for multigene expression. This review describes some of the techniques that have been used to express multiple genes, and will focus on the use of 2A peptides or 2A peptide-like sequences in the design of multicistronic vectors that may alleviate some of these issues.
Assuntos
Vetores Genéticos/genética , Peptídeos/genética , Tecnologia Farmacêutica/métodos , Sequência de Aminoácidos/genética , Animais , Vetores Genéticos/síntese química , Humanos , Dados de Sequência Molecular , Peptídeos/síntese químicaRESUMO
Attempts to generate reliable and versatile vectors for gene therapy and biomedical research that express multiple genes have met with limited success. Here we used Picornavirus 'self-cleaving' 2A peptides, or 2A-like sequences from other viruses, to generate multicistronic retroviral vectors with efficient translation of four cistrons. Using the T-cell receptor:CD3 complex as a test system, we show that a single 2A peptide-linked retroviral vector can be used to generate all four CD3 proteins (CD3epsilon, gamma, delta, zeta), and restore T-cell development and function in CD3-deficient mice. We also show complete 2A peptide-mediated 'cleavage' and stoichiometric production of two fluorescent proteins using a fluorescence resonance energy transfer-based system in multiple cell types including blood, thymus, spleen, bone marrow and early stem cell progenitors.