Construction And Identification Of Lentiviral Vectors With Overexpression CTTN

The CTTN gene is a thin,elongated monomer,located on human chromosome 11q13.3,and its encoded cortactin containing 550 amino acids is present in all cell types.Cortactin contains NTA domain,Repeat domain,Helix domain,PRD domain and SH3 domain.The structure of cortactin is highly conserved among all species.Cortactin can promote the polymerization and rearrangement of the actin cytoskeleton,and plays an important role in the formation of cellular pseudopodia,the formation of invasive pseudopodia,cell migration and endocytosis.The most common apparent modifications of cortactin are tyrosine phosphorylation and serine/threonine phosphorylation,and the mechanism is clear.In addition to phosphorylation,cortactin can also be acetylated,but few studies have been conducted and the mechanism remains unclear.In this study,we constructed the CTV lentivirus expression vector pGV358-CTTN.After validation by PCR and sequencing,293 T cells were co-transfected with lentivirus packaging plasmids pHelper 1.0 and pHelper 2.0 to package lentiviruses.Lentivirus was collected by ultracentrifugation and the obtained concentrated lentivirus was identified by quality.Mycoplasma,chlamydia,bacteria,and fungi were all negative and endotoxin content was <50 EU/mL.The lentiviral titer of overexpression of CTTN was 2E+8TU/mL by fluorescence counting.The construction of CTTN-overexpressing lentivirus provides an experimental basis for studying the acetylation of cortactin.