1993 Fiscal Year Final Research Report Summary
Development of cloning methods for cultured insect cells
Grant-in-Aid for General Scientific Research (B)
|Allocation Type||Single-year Grants |
|Research Institution||Tokyo University of Agriculture & Technology |
MITSUHASHI Jun Tokyo University of Agriculture and Technology Faculty of Agriculture, Professor, 農学部, 教授 (90209809)
IWABUCHI Kikuo Tokyo University of Agriculture and Technology Faculty of Agriculture, Professor, 農学部, 助教授 (00203399)
KUNIMI Yasuhisa Tokyo University of Agriculture and Technology Faculty of Agriculture, Professor, 農学部, 助教授 (50195476)
HUKUHARA Toshihiko Tokyo University of Agriculture and Technology Faculty of Agriculture, Professor, 農学部, 教授 (70011880)
|Project Period (FY)
1992 – 1993
|Keywords||Cell cloning / Cultured insect cells / Conditioned media / Cloning media / Cloning vessels|
Development of widely applicable cloning method for cultured insect cells was aimed because insect cell cloning was possible only in limited cell lines.
1.Isolation method for single cells : A single cell could be isolated by the following method. Diluted cell suspension was made in a single cell culture medium. From the cell suspension, a single cell was aseptically sucked into a glass capillary with a tip of 20mum in diameter, under an inverted microscope. This method was effcint enough for routine application.
2.Single cell culture vessels : In order to supply growth promoting substance(s) liberated from cultured cells to the isolated single cell, a small compartment was made by gluing a cylinder made with a membrance filter to the bottom of a culture dish. By placing a single cell into the cylinder and culturing mother cell population outside the cylinder, the single cell grew to some extent.
3.Solid medium for growing a single cell : An agarose, Seaplaque agar was found to be usable.
In the medium containing 0.2% agarose, Survivorship, percentage of colony formation, ratio of cell division of the single cell were better, but a clone could not be obtained.
4.Conditioned medium for culturing single cell ; The medium conditioned for 7-10 days with homologuous cells was found suitable. In the medium consisted of equal volume of the conditioned medium and a nutrient rich MGM-450 medium, most of isolated cells could multiply. However, a cell line from the mulberry tiger moth could not be cloned.
5.Characterization of clones : It became evident that a clone became heterogeneous cell population soon after the cloning. Variations inshape, size and chromosome number were wimilar to those of the mother cell population. Growth ratio of clones were also similar to that of their original cell lines.
6.Susceptibility of clones to viruses : Among clones isolated from common armyworm cell line, some showed higer susceptiblilty to Ivela aurepes NPV than original cell line.
7.Storage of cloned cells : Isolated clones could be freeze-stored at -80ﾟC by adding 10% glycerol to the cell suspension. Less