Gene modification via "plug and socket" gene targeting

Jada Lewis; Baoli Yang; Pete Detloff; Oliver Smithies

doi:10.1172/JCI118403

Conventional gene targeting experiments in embryonic stem (ES) cells frequently involve the disruption of endogenous sequences with a positively selectable marker such as neomycin phosphotransferase (neo) or hypoxanthine phosphoribosyl-transferase (HPRT), often in conjunction with a negative selectable marker such as herpes simplex thymidine kinase. ES cells which have incorporated the positive marker and have lost the negative marker, as determined by selection with the appropriate drugs, can be further analyzed by Southern hybridization or PCR to identify cell lines that have been correctly targeted. Repeatedly altering a chosen target gene, in order, for example, to study the effects of a series of related but not identical changes, can, however, be very time consuming unless the target gene is easily targeted. Strategies have therefore been devised to allow easier isolation of targeted cells when such repeated modifications are desired. Two-step gene targeting is such a strategy. The first targeting step introduces a selectable market into the genome of the ES cells by conventional gene targeting. The second targeting step in several versions of the two-step strategy has used a series of different constructs to obtain the desired modifications, while at the same time deleting the marker which was inserted in the first step. This second event is therefore detected by selecting against the initial marker gene. Deletion is not, however, the only mechanism by which a marker gene function can be lost. For example, spontaneous mutations can produce a nonfunctional marker gene. Such mutations therefore give rise to a "background" of nonrecombinant colonies, and force additional screening to eliminate colonies which should have been excluded during the second selection. Although the spontaneous mutational event may itself be infrequent, if it occurs in culture before the targeted loss of the marker gene, the background of nontargeted clones may be considerable. Termed the "fluctuation phenomenon" because its magnitude fluctuates with the time of its occurrence, the problem can be eliminated by using positive selection for both the first and second gene targeting events. We have devised a version of the two-step targeting strategy, termed the "plug and socket" method, which can be used repeatedly to make subtle mutations or to replace entire genes while avoiding the problem of background colonies by using only positive selection. This method in its present form can be used to target any murine gene in HPRT deficient ES cells.

Gene modification via "plug and socket" gene targeting

Files and links (1)

Abstract

Details

Metrics