Gene targeting in Arabidopsis

Abstract

SummaryPrecise modi®cation by gene targeting (GT) provides an important tool for studies of gene function in vivo. Although routine with many organisms, only isolated examples of GT events have been reported for¯owering plants. These were at low frequencies precluding reliable estimation of targeting ef®ciency and evaluation of GT mechanisms. Here we present an unambiguous and straightforward system for detection of GT events in Arabidopsis using an endogenous nuclear gene encoding protoporphyrinogen oxidase (PPO), involved in chlorophyll and heme syntheses. Inhibition of PPO by the herbicide Butafenacil results in rapid plant death. However, the combination of two particular mutations renders PPO highly resistant to Butafenacil. We exploited this feature for selection of GT events by introducing the mutations into the PPO gene by homologous recombination. We have estimated the basal GT frequency to be 2.4 Q 10 ±3 . Approximately one-third of events were true GT (TGT) leading to the anticipated modi®cation of the chromosomal PPO copy. The remaining events could be classi®ed as ectopic GT (EGT) arising by modi®cation of vector DNA by the chromosomal template and its random integration into the Arabidopsis genome. Thus the TGT frequency in our experimental setup is 0.72 Q 10 ±3 . In view of the high ef®ciency of Arabidopsis transformation, GT experiments of a reasonable size followed by a PCR screen for GT events should also allow for modi®cation of non-selectable targets. Moreover, the system presented here should contribute signi®cantly to future improvement of GT technology in plants.

Site-directed mutagenesis in Arabidopsis using custom-designed zinc finger nucleases

“…Efficient GT procedures have been available for more than 20 y in yeast (1) and mouse (2). Successful GT has also been achieved in Arabidopsis and rice plants (3)(4)(5)(6). Typically, GT events occur in a fairly small proportion of treated mammalian cells (approximately 1% of the total random integration events in mouse ES cells).…”

Targeted mutagenesis using zinc-finger nucleases in Arabidopsis

“…More recently, enhanced GT efficiencies were reported by using two alternative strategies. In the first study, DNA was introduced into Arabidopsis plants by using the vacuum-infiltration method of Agrobacterium-mediated transformation (22), resulting in targeting ratios of 7.2 ϫ 10 Ϫ4 GT events per integration (23). In the second strategy, the homologous sequence was flanked on both sides with negativeselectable marker genes and introduced into cultured rice cells by using Agrobacterium-mediated transformation; although the targeting ratio was low (6.5 ϫ 10 Ϫ4 GT events per integration), the stringent negative-selection scheme eliminated most random integrations, and Ϸ1% of survivors were the result of targeting events (24).…”

Nonhomologous End Joining-Mediated Gene Replacement in Plant Cells

“…Thus, for example, HR-mediated gene targeting has been enhanced in Arabidopsis (Arabidopsis thaliana) plants by overexpression of RAD54, a yeast chromatin-remodeling protein (Shaked et al, 2005). Other examples include the use of strong positive-and negative-selection schemes or PCR-based screening for the selection of rare HR-mediated genetargeting events in rice (Oryza sativa; Terada et al, 2002Terada et al, , 2007 and in Arabidopsis plants and tissues (Kempin et al, 1997;Hanin et al, 2001), respectively. While these approaches and others have proven useful, relying on the cell's natural low rate of HR DNA repair has shown only limited success in the targeting of native and transgenic sequences in plant cells (for review, see Puchta, 2002;Hanin and Paszkowski, 2003;Reiss, 2003;Porteus, 2009;Weinthal et al, 2010;Tzfira et al, 2012).…”