SUMMARY The main bottleneck in the application of biotechnological breeding methods to woody species is due to the in vitro regeneration recalcitrance shown by several genotypes. On the other side, woody species, especially grapevine ( Vitis vinifera L.), use most of the pesticides and other expensive inputs in agriculture, making the development of efficient approaches of genetic improvement absolutely urgent. Genome editing is an extremely promising technique particularly for wine grape genotypes, as it allows to modify the desired gene in a single step, preserving all the quality traits selected and appreciated in elite varieties. A genome editing and regeneration protocol for the production of transgene‐free grapevine plants, exploiting the lipofectamine‐mediated direct delivery of CRISPR–Cas9 ribonucleoproteins (RNPs) to target the phytoene desaturase gene, is reported. We focused on Nebbiolo ( V. vinifera ), an extremely in vitro recalcitrant wine genotype used to produce outstanding wines, such as Barolo and Barbaresco. The use of the PEG‐mediated editing method available in literature and employed for highly embryogenic grapevine genotypes did not allow the proper embryo development in the recalcitrant Nebbiolo. Lipofectamines, on the contrary, did not have a negative impact on protoplast viability and plant regeneration, leading to the obtainment of fully developed edited plants after about 5 months from the transfection. Our work represents one of the first examples of lipofectamine use for delivering editing reagents in plant protoplasts. The important result achieved for the wine grape genotype breeding could be extended to other important wine grape varieties and recalcitrant woody species.
Targeting tyrosine kinase receptors (RTKs) with specific Abs is a promising therapeutic approach for cancer treatment, although the molecular mechanism(s) responsible for the Abs’ biological activity are not completely known. We targeted the transmembrane RTK for hepatocyte growth factor (HGF) with a monoclonal Ab (DN30). In vitro , chronic treatment of carcinoma cell lines resulted in impairment of HGF-induced signal transduction, anchorage-independent growth, and invasiveness. In vivo , administration of DN30 inhibited growth and metastatic spread to the lung of neoplastic cells s.c. transplanted into immunodeficient nu / nu mice. This Ab efficiently down-regulates HGF receptor through a molecular mechanism involving a double proteolytic cleavage: ( i ) cleavage of the extracellular portion, resulting in “shedding” of the ectodomain, and ( ii ) cleavage of the intracellular domain, which is rapidly degraded by the proteasome. Interestingly, the “decoy effect” generated by the shed ectodomain, acting as a dominant negative molecule, enhanced the inhibitory effect of the Ab.
