Replacing an endogenous with exogenous gene in a stable cell line

Gregory O'Sullivan g.osullivan at
Fri Sep 18 14:40:45 EST 1998

I am looking for advice on how best to proceed with the following problem.
I am trying to block the expression of an endogenous gene and subsequently
reintroduce it in an altered form. I have a plan of action but am not quite
sure if stands a realistic chance of succeeding.
	The first aim would be to knockout endogenous expression by creating a
stable cell line in which the antisense sequence of one of the introns
within my gene is expressed under the control of the inducible MMTV promoter.
	The next stage would be to express the coding sequence of my gene (wild
type, mutated, or tagged - all of which are at my disposal) under the
control of another inducible vector such as tetracycline in the knockout
cell line.
	The issue is further complicated by the fact that my cell will then be
differentiated for several weeks in order to produce mature neuron like
cells. Thus, I am concerned about the long term toxicity of these agents as
well as the possible loss of one or both of the vectors over time.
	Any suggestions or comments good, bad or indifferent would be most

Gregory O'Sullivan,
Neurobiology group,
Department of Biochemistry,
Imperial College,
London SW7 2AY,

email: G.Osullivan at
Tel: (44) 0171 5895111 ext 55203

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