AAV infection of bovine embryos: Novel, simple and effective tool for genome editing.

Adeno-associated viruses (AAV) are widely used in the field of genetically modified organism production. In this work, transduction of bovine embryos by AAV was selected as a potential approach to perform genetic modifications: we have used recombinant AAV to produce GFP-positive bovine embryos. Five different AAV serotypes were used to evaluate their ability to deliver genetic material into the bovine embryos. AAV9 serotype demonstrated minimal effectiveness (38,10%) as the genetic material transfer tool. Four other serotypes of AAVs (AAV1, AAV2, AAV6 and AAV-DJ) showed very close transduction efficiency (52,94-58,33%). CD209 is a C-type lectin receptor which is presented on the surface of macrophages and dendritic cells. CD209 recognizes a broad range of pathogens in a rather nonspecific manner. Production of CD209 knock-out is relevant for better understanding of infection mechanisms. Potentially, production of such knock-out may enable animals to become resistant to various infections. We have analyzed DNA samples from 22 blastocysts obtained after in vitro culture of zygotes subjected to recombinant AAV action. We have detected that 3 of 22 analyzed blastocysts contained mosaic CD209 frameshifts. Therefore, we have demonstrated proof of principle that application of AAV as a genome editing tool is an effective method for obtaining genetically modified cattle embryos.

Design and Analysis of a Mutant form of the Ice-Binding Protein from Choristoneura fumiferana.

Ice-binding proteins are expressed in the cells of some cold adapted organisms, helping them to survive at extremely low temperatures. One of the problems in studying such proteins is the difficulty of their isolation and purification. For example, eight cysteine residues in the cfAF (antifreeze protein from the eastern spruce budworm Choristoneura fumiferana) form intermolecular bridges during the overexpression of this protein. This impedes the process of the protein purification dramatically. To overcome this issue, in this work, we designed a mutant form of the ice-binding protein cfAFP, which is much easier to isolate that the wild-type protein. The mutant form named mIBP83 did not lose the ability to bind to ice surface. Besides, observation of the processes of freezing and melting of ice in the presence of mIBP83 showed that this protein affects the process of ice melting, increasing its melting temperature, and does not decrease the water freezing temperature.