In vitro fusion

Pless DD, Wellner RB. In vitro fusion of endocytic vesicles effects of reagents that alter endosomal pH. J Cell Biochem 1996 62(l) 27-39. 

The production in vitro of mAbs with a predetermined specificity has only been possible since the advent of the technology of hybridomas, which was introduced by Kohler and Milstein in 1975. These hybridomas are the products of in vitro fusion of myelomas with normal B lymphocytes. The fusion products preserve the capacity for self-propagation in a culture, as well as the secretion of the antibodies of interest, characteristics inherited from the parent myeloma and the normal B lymphocyte, respectively. The myelomas used in such fusions generally involve cell lines from B-lymphocyte tumors developed in mice or rats (Cotton and Milstein, 1973 Kohler and Milstein, 1975b Shulman et al., 1978), while... 

Somatic cell hybridization The in vitro fusion of animal cells derived from somatic cells that differ genetically. 

Betthouser et al. In vitro Fusion lonomycin and 6-di-methyl-amino-purine Fetal fibroblast (3 passages) genital ridge (0 passage) 143 164 8 hours (one cell stage) 72 hours (4 cell stage) Confluent no serum starvation... 

Fig. 1. Principle of the in vitro fusion assay. After labeling the endocytic pathway with acridinium-transferrin (Ac-Tfn) labeled endosomes (donor) are enriched by density centrifugation. Recycling endosomes (acceptor) are purilied from unlabeled cells first by density centrifugation followed by immunoisolation using anti-Rabll coated magnetic beads. Transport of Ac-Tfn from the donor endosomes to acceptor recycling endosomes is performed at 3T and in the presence of cytosol and ATP. After washing the beads-bound membranes, transferred Ac-Tfn is measured by detecting acridinium cleavage in a luminometer.

The combined methods of preparing an endosome-enriched fraction labeled with acridinium-transferrin and specific immunoadsorption of unlabeled Rabll-positive recycling endosomes provide the basis of our in vitro fusion assay. Using this assay, transport of the physiological marker transferrin from labeled donor endosomes to immunoisolated acceptor recycling endosomes can be followed by measuring arrival of acridinium-transferrin to beads-bound endosomes as detected by lashlight-luminescence in a luminometer. 

The in vitro fusion approach, which is most commonly used, has two obvious advantages (i) the possibility for direct and precise controls over the process of gene fusion and (ii) the availability of an assortment of general and specialized vectors. 

The surface potential can play an important role in the behavior of liposomes in vivo and in vitro (e.g.. Senior, 1987). In general, charged liposomes ai e more stable against aggregation and fusion than uncharged vesicles. However, physically stable neutral liposomes have been described (e.g.. Van Dalen et al., 1988). They are sufficiently stabilized by repulsive hydration forces, which counteract the attractive van der Waals forces. 

Human insulin Two peptide chains A, 21 amino acids long, and B, 30 amino acids long . coli Juvenile onset diabetes Approved for sale A and B chains made separately as fusion proteins and joined in vitro Compared with animal Insulins some undesirable side-effects have been noted... 

Schilling, M., F. Patett et al. (2007). Influence of solubility-enhancing fusion proteins and organic solvents on the in vitro biocatalytic performance of the carotenoid cleavage dioxygenase AtCCDl in a micellar reaction system. Appl. Microbiol. Biotechnol. 75(4) 829-836. 

Kahn TW, Beachy RN, Falk MM (1997) Cell-free expression of a GFP fusion protein allows quantitation in vitro and in vivo. Curr Biol 7 R207-R208... 

Roberts, R.W. and Szostak, J.W. (1997) RNA-peptide fusions for the in vitro selection of peptides and proteins. Proceedings of the National Academy of Sciences of the United States of America, 94, 12297-12302. 

---