Twenty Years of Gene Targeting
What We Don't Know
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It has been a little over 20 years since targeted gene disruption in the mouse was reported. This Perspectives article examines how we got to where we are and paints in broad strokes what we have and have not learned.
Homologous recombination was well known in the latter half of the 20th century,1,2 but its use in the directed mutation, disruption, and alteration of the mammalian genome was hampered by the vanishingly small frequency with which it occurred.3 However, with powerful selection techniques, these infrequent events could be detected, and in 1987 to 1988 the Smithies and Capecchi laboratories published data documenting general strategies for targeted disruption in mouse embryo–derived stem cells.4–6 As targeted gene disruption of the mouse genome became established, transcription factors that impacted organ development were first, obvious targets, and because mouse embryo viability is dependent on a functional heart beginning at around day 10.5 to 11 of development, the essential role(s) these factors played in cardiac development began to become apparent.7
Scientific advances often come about because of a fortuitous mix of technology, people, and circumstance, and such was the case for a first, seminal study in which a gene known to be vital in controlling cardiac contractility was targeted for deletion. Thomas Doetschman, the first author on the paper from the Smithies' laboratory, was recruited to the University of Cincinnati, where Evangelia Kranias was working on the small protein, phospholamban. Kranias initiated a minisabbatical with Doetschman, taking on the project to “knockout” the phospholamban gene. The paper, which was published in Circulation Research in 1994,8 not only helped to define the role phospholamban played in normal cardiac function but also brought home to the cardiovascular community the powerful approach represented …