Cloning

Cells derived from a single cells through mitosis constitute a clone and process of obtaining clonies is called cloning. (A sexual progeny of single individual make up a clone). In simple terms cloning consists of trypsinisation of a monolayer culture to prepare a cell suspension 3-4 dilution steps to achieve suitable cell density (10-200cells/ml) and seeding in petri dishes or flasks or multiwell dishes the culture vessel are incubated for 1-3 weeks with a medium change after 1 week by this time colonies will develop.
Colonies may be isolated (1) directly from multiwell dishes by tryupsinisation of usually such wells which contained only one cell the start or which have single distinct colony originating from single cell lying away from other nondividing from petriplates is done by using cloning ringes which are placed around the desired colonies after the medium is poured. The cell colony within each porclain teflon well of multiwell plate or in a flask. Alternatively (3) the desired colony may shielded and the remaining colonies are irridiated by a lethal dose (3,000rads) The protected colony is trypsinised and the cells are cloned in the same plate the irridiated cells serving as a feeder layer.
plating efficiency (per cent of cells forming colonies of continuous cell lines is generally 10% or higher, while that of finite cell lines may be quite low say 0.5- 5% or even zero several approaches have been used to enhance the plating efficiency e.g (1) use of a rich medium (2) use of serum espacially foetal calf serum in the medium (3) using conditioned medium (4) use of feeder layer (5) addition of hormones like insulin dexamethasone etc. (6) providing intermediate metabolites like keto acids, nucleosides etc.
A feeder layer is a layer of cells which have been treated to prevent their growth and ultimately cause their death these cells however, provied the necessary metabolites to enahance the plating embryo fibroblast from primary culture and reseeding the cells at density. When the monolayer reaches 50% confluence is covered by the monolayer the cells are either treated with mitomycin overnight or are irridiated with and X-rays. After treatemnet the cells are incubated for 24 hours in fresh medium trypsiised and reseded at and incubated for 24-48 hours. This yields the feeder layer on which cells to be cloned are seeded incubated and desired colonies isolated. Feeder layer may be established using homologous cells but is preferable to use heterologous cells for easy deetection of accidental cross contamination in the isolated clones.
Cloning is used to (1) obtain homogeneous cell lines from heterogeneous cell (2) to isolate biochemical mutants and (3) cell strains with marker chromosomes and (4) to develop hybridoma clones. Cloning is generally applied to continuous cell lines but often their clones become considerable heterogenous by the time they are sufficient multiplied for use. The problem with finite cell lines is that of life span, by the time the clone is suffi