Cloning

Cells derived from a single cell through tmitosis constitute a clone and the process of obtaining clones is called cloning. In simple term cloning consists of trypsinisation of a monl=olayer culture to prepare a cell suspension, 3-4 dilution steps to achieve a suitable cell density (10- 200 cell/ml) and seeding in petri dishes or flasks or multi well dishes The culture vessel are incubated for 1-3 weeks with a medium change after 1 week by this time colonies will develop.
Colonies a may be isolated (1) Directly from multiwell dishes by trypsinisation of usually such wells which contained only one cell at the start, or which have single distinct colony origination from a single cell lying away from other nondividing cell or cells. This is confirmed by microscopic observation (2) Isolation of clones from petriplates is done by using cloning rings which ar eplaced around the desired colonies after the medium is poured. The cell colony within each porcelain teflon or stainless steel ring is trypsinized, cell are suspended in medium and seeded in a shielded and the remaining colonies are irridiated by a lethal dose The protected colony is trypsinised and the cells are cloned in the same plate, the irridiated cells serving as a feeder layer.

Plating efficiency lines is generally 10% or higher, wehile that of finiite cell lines may be quite low say 0.5-5% or even zero. Several approaches have been used to enhance the planting efficiency e.g (1) use of rich medium, (2) use of serum, especially foetal cell serum, in the medium, (3) using conditioned medium grwon for a period of time (4) use of feeder layer, (5) addition of hormones like insulin, dexamethason etc, (6) providing intermediate metabolited like keto acids nucleosides etc.