Baculovirus-mediated gene transfer into pancreatic islet cells.

Abstract

Baculovirus transduction is a gene transfer method that uses a moth cell virus for mammalian cells in culture, which results in a high-level prolonged expression. Here we demonstrate that recombinant baculoviruses can serve as efficient gene transfer vehicles for delivering foreign genes driven by mammalian promoters into human and mouse pancreatic islet cells. Existing methods, such as various transfection and electroporation techniques, either suffer from low efficiency or cause extensive membrane damage. Viral vectors have emerged as an important tool for gene delivery and expression in mammalian cells but suffer from several drawbacks, such as lengthy construction time and expression of viral genes. The baculovirus Autographa californica multiple nuclear polyhedrosis virus is widely used as a vector for expression of foreign genes in insect cells and, more recently, in some mammalian cells. Using several green fluorescent protein- and LacZ-expressing constructs in a cytomegalovirus promoter cassette, we obtained efficient gene expression in primary human and mouse islet cells. There was no impairment of glucose-stimulated intracellular free calcium responses after baculovirus infection. The safety and the relative ease of construction and propagation of the virus makes the baculovirus system a useful tool for facilitating the transfer of foreign genes.

Baculoviruses exhibit restricted cell type specificity in rat brain: a comparison of baculovirus- and adenovirus-mediated intracerebral gene transfer in vivo

“…Previously, it has been shown that they possess the capacity to transduce a variety of mammalian cell types including mesenchymal cells, neural glial cells and hepatocytes. [1][2][3][4][14][15][16][17] Our results show that baculoviruses are able to transduce brain cells efficiently in vivo and possess a very specific cell tropism in rat brain. The choroid plexus epithelial cells were transduced with high efficiency (Figure 1, Table 1).…”

Baculovirus Entry into Human Hepatoma Cells

“…So far, baculovirus vectors have been shown to transduce various dividing and nondividing mammalian cell lines in vitro (1,8,12,20,29,42,48,49,52). Baculovirus-mediated gene transfer has also been successful in vivo (1,21,23,24,27,42,47,48,50).…”

Direct imaging shows that insulin granule exocytosis occurs by complete vesicle fusion

“…Data were collected at 1.3-1.7 Hz because most vesicle fusion events appeared to last several seconds. Cells and islets were superfused with Krebs-Ringer buffer containing 119 mM NaCl, 4.7 mM KCl, 25 mM NaHCO 3 , 2.5 mM CaCl 2 , 1.2 mM MgSO 4 , 1.2 mM KH 2 PO 4 , and 25 mM glucose (MIN6 cells) or 11 mM (beta cells or islets) (17). All imaging experiments were conducted at 35-37°C.…”

“…Islet cells were dissociated by using 0.25 mg͞ml trypsin. Cells or islets were cultured on glass coverslips and incubated in RPMI medium 1640 containing 11.5 mM glucose, 10% FBS, 100 units͞ml penicillin, and 100 g͞ml streptomycin at 37°C for 1-2 days before transduction (17). MIN6 cells (18) were grown in DMEM (Life Technologies) containing 25 mM glucose, 10% FBS, 100 units͞ml penicillin, and 100 g͞ml streptomycin.…”