Construção de um gene repórter de membrana usando sequência do tipo strep-tag

Abstract

Gene transfer is a biotechnological tool widely used in cell and molecular biology studies. This methodology is based on the introduction of exogenous genes into target cells, using vectors carrying this genetic material as mediators. Because cellular transformation does not reach all the cells, it is necessary to separate the transformed cells from those not transformed, using their physical, chemical or biological characteristics. Currently, two mechanisms are used to carry out such processes, one of them is the selection by antibiotics resitance. However, this procedure is time consuming, difficult to use the methodology as affordable selection of technics. The other method is based on the use of fluorescent reporter genes and subsequent isolation by cells sorting technique using flow cytometry. However, not all laboratories have this equipment, due to its high cost. The reporter gene developed in the present work, named as L7, encodes a chimeric protein formed by a protein transmembrane domain and a nine amino acid extracellular domain with high affinity for streptavidin. The gene thus formed has an approximate size of 300 pb providing a large potential for use in gene transfer experiments.