Gene transfer into mammalian cells using a self-deleting avian leukemia and sarcoma virus-based vector.

Objectives: We have previously described an avian leukemia and sarcoma virus-based vector containing an additional att sequence in an internal position that is capable of self-deleting most of its 5' viral sequences during one cycle of replication in avian cells [Virus Res 2008;135:72-82; Arch Virol 2008;153:2233-2243]. Herein, our aim was to test the infectivity and self-deleting properties of this avian retroviral vector in human cells.

Methods: Human Hela cells transiently expressing the cellular receptor for avian leukemia and sarcoma viruses (tva) were infected with the avian vector.

An improved self-deleting retroviral vector derived from avian leukemia and sarcoma virus.

We have previously developed a self-deleting avian leukosis and sarcoma virus (ALSV)- based retroviral vector carrying an additional attachment (att) sequence. Resulting proviruses underwent deletion of viral sequences and were flanked either by two LTRs (LTRs proviruses) or by the additional att sequence and the 3' LTR (att proviruses). Herein, we have tried to increase (1) the self-deleting properties of this vector, either by raising the selection pressure applied on target cells or by optimizing the size of the internal att sequence, (2) the titer of the vector by deleting or inverting some viral sequences.

A novel self-deleting retroviral vector carrying an additional sequence recognized by the viral integrase (IN).

During retroviral integration, the viral integrase recognizes the attachment (att) sequence (formed by juxtaposition of two LTRs ends) as the substrate of integration. We have developed a self-deleting Avian Leukosis and Sarcoma Viruses (ALSVs)-based retroviral vector carrying an additional copy of the att sequence, between neo and puro genes. We observed that: (i) the resulting NP3Catt vector was produced at neo and puro titers respectively smaller and higher than that of the parental vector devoid of the att sequence; (ii) 61% of NP3Catt proviruses were flanked by LTRs; most of them were deleted of internal sequences, probably during the reverse transcription step; (iii) 31% of clones were deleted of the whole 5' part of their genome and were flanked, in 5', by the additional att sequence and, in 3', by an LTR.

High-affinity uranyl-specific antibodies suitable for cellular imaging.

Monoclonal antibodies (mAbs) have proved to be valuable models for the study of protein-metal interactions, and previous reports have described very specific antibodies to chelated metal ions, including uranyl. We raised specific mAbs against UO2(2+)-DCP-BSA (DCP, 1,10-phenanthroline-2,9-dicarboxylic acid) to generate new sets of antibodies that might cross-react with various complexed forms of uranyl in different environments for further application in the field of toxicology. Using counter-screening with UO2(2+)-DCP-casein, we selected two highly specific mAbs against uranyl-DCP ( K D 10-100 pM): U04S and U08S.

Structural consequences of binding of UO2(2+) to apotransferrin: can this protein account for entry of uranium into human cells?

It has been established that transferrin binds a variety of metals. These include toxic uranyl ions which form rather stable uranyl-transferrin derivatives. We determined the extent to which the iron binding sites might accommodate the peculiar topographic profile of the uranyl ion and the consequences of its binding on protein conformation.