31 Cell Cloning
317
Fig. 3. Plate devised for cloning cell lines. The hatched area represents the culture
medium. The cloning plate consists of the base of a 3-cm petri dish (3), which was
attached to the underside of a 5-cm petri dish lid (5). The feeder layer (F) and
clones (C) are also indicated. Reproduced from Peel, D.J. and Milner,M.J. (1990),
"The diversity of cell morphology in cloned cell lines derived from Drosophila
imaginal discs", Roux's Archives of Developmental Biology, Vo1.198, 479-482, Fig.l,
1990, with permission of copyright owner, Springer-Verlag
Comments
Substrate-dependent cells should be used as the cell line used to make
the feeder layer. It is not necessary to use homologous cells as feeder
layer cells.
More than one cell can be seeded on a feeder layer in one petri dish if
the cells make compact cell colonies. However, in this case, repetition of
the cloning procedure twice or more is preferable.
In general, insect cells are said to be able to recover from irradiation
damage. Therefore, it is important to determine the conditions of irradiation treatment. Tolerance to irradiation with X-rays and y-rays is different at different developmental stages. In insects, younger stages are
more sensitive to irradiation than older stages. For example, the lethal
dose of irradiation for insects is said to be more than 2.58 x 10- 2 c/kg in
the embryonic stage, several 0.258 c/kg in the larval stage, several 2.58 c/
kg in the pupal stage and more than 25.8 c/kg in the imaginal stage (compared with 10.32 x 10- 2 c/kg for humans). Then, the stage from which the
cell line originates must be considered.
Addendum
For substrate-dependent cells, a feeder layer of non-irradiated cells may
be used according to the methods of Peel and Milner (1990). Their method
is as follows.
Procedure
1. The cloning dish is made by attaching an inverted 3-cm plastic petri
dish base to the underside of the lid of a 5-cm plastic petri dish (Fig. 3).
The two are joined together after dipping the rim of the smaller dish
into propylene oxide, which partly dissolves the plastic.
1. Seed approximately 3 x 10 6 feeder layer cells into a 5-cm petri dish.
2. Culture the feeder layer cells for 2-3 days before use.
3. Prepare several feeder layer dishes.
Preparation of
cloning dish
Feeder
layer
formation
317
Fig. 3. Plate devised for cloning cell lines. The hatched area represents the culture
medium. The cloning plate consists of the base of a 3-cm petri dish (3), which was
attached to the underside of a 5-cm petri dish lid (5). The feeder layer (F) and
clones (C) are also indicated. Reproduced from Peel, D.J. and Milner,M.J. (1990),
"The diversity of cell morphology in cloned cell lines derived from Drosophila
imaginal discs", Roux's Archives of Developmental Biology, Vo1.198, 479-482, Fig.l,
1990, with permission of copyright owner, Springer-Verlag
Comments
Substrate-dependent cells should be used as the cell line used to make
the feeder layer. It is not necessary to use homologous cells as feeder
layer cells.
More than one cell can be seeded on a feeder layer in one petri dish if
the cells make compact cell colonies. However, in this case, repetition of
the cloning procedure twice or more is preferable.
In general, insect cells are said to be able to recover from irradiation
damage. Therefore, it is important to determine the conditions of irradiation treatment. Tolerance to irradiation with X-rays and y-rays is different at different developmental stages. In insects, younger stages are
more sensitive to irradiation than older stages. For example, the lethal
dose of irradiation for insects is said to be more than 2.58 x 10- 2 c/kg in
the embryonic stage, several 0.258 c/kg in the larval stage, several 2.58 c/
kg in the pupal stage and more than 25.8 c/kg in the imaginal stage (compared with 10.32 x 10- 2 c/kg for humans). Then, the stage from which the
cell line originates must be considered.
Addendum
For substrate-dependent cells, a feeder layer of non-irradiated cells may
be used according to the methods of Peel and Milner (1990). Their method
is as follows.
Procedure
1. The cloning dish is made by attaching an inverted 3-cm plastic petri
dish base to the underside of the lid of a 5-cm plastic petri dish (Fig. 3).
The two are joined together after dipping the rim of the smaller dish
into propylene oxide, which partly dissolves the plastic.
1. Seed approximately 3 x 10 6 feeder layer cells into a 5-cm petri dish.
2. Culture the feeder layer cells for 2-3 days before use.
3. Prepare several feeder layer dishes.
Preparation of
cloning dish
Feeder
layer
formation