Gene replacement

Morton, R.; Hooykaas, Paul J. J.

doi:10.1007/bf01249697

Cited by 27 publications

(10 citation statements)

References 81 publications

(95 reference statements)

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“…Homologous recombination ("gene targeting") is an attractive technique for integrating foreign DNA at a specific site in the mouse genome (44). Unfortunately, no attractive technique for targeting of genes of higher plants could yet be developed (45). As the induction of DSBs by I-Sce I seems to be rate-limiting in our system (Table 1), the frequency of integration, which was in the best case one homologous event out of '50 illegitimate ones, might be further improved by, for example, supplying more enzyme directly (46,47).…”

Section: Discussionmentioning

confidence: 99%

Two different but related mechanisms are used in plants for the repair of genomic double-strand breaks by homologous recombination.

Puchta

Dujon

Höhn

1996

Proc. Natl. Acad. Sci. U.S.A.

361

283

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Genomic double-strand breaks (DSBs) are key intermediates in recombination reactions of living organisms. We studied the repair of genomic DSBs by homologous sequences in plants. Tobacco plants containing a site for the highly specific restriction enzyme I-Sce I were cotransformed with Agrobacterium strains carrying sequences homologous to the transgene locus and, separately, containing the gene coding for the enzyme. We show that the induction of a DSB can increase the frequency of homologous recombination at a specific locus by up to two orders of magnitude. Analysis of the recombination products demonstrates that a DSB can be repaired via homologous recombination by at least two different but related pathways. In the major pathway, homologies on both sides of the DSB are used, analogous to the conservative DSB repair model originally proposed for meiotic recombination in yeast. Homologous recombination of the minor pathway is restricted to one side ofthe DSB as described by the nonconservative one-sided invasion model. The sequence of the recombination partners was absolutely conserved in two cases, whereas in a third case, a deletion of 14 bp had occurred, probably due to DNA polymerase slippage during the copy process. The induction of DSB breaks to enhance homologous recombination can be applied for a variety of approaches of plant genome manipulation. (7), which has an 18-bp recognition site (8), was used to induce recombination reactions. In previous studies performed with I-Sce I, specific DSBs were induced in vivo in plasmid DNA transfected or injected into eukaryotic cells (9-12). Recently I-Sce I-mediated induction of genomic DSBs and their repair by homologous recombination were described for mouse cells (13,14). We demonstrated that expression of the I-Sce I gene led to specific DSBs in vivo in DNA molecules transfected into plant protoplasts (10). As a consequence extrachromosomal homologous recombination was induced. This reaction proceeds mainly via single-strand annealing and thus seems to be different from recombination reactions in the chromosome (as discussed in ref. 20). In the present communication we analyzed the mechanism by which DSBs at specific sites in the plant genome are repaired with homologous sequence information, and we tested whether induction of DSBs is correlated with an increase in recombination frequencies. MATERIALS AND METHODSCloning Procedures. Construction of the I-Sce I expression vector. The plasmid p35SISceI+ (10) containing an artificial I-Sce I open reading frame (ORF) under the control of the cauliflower 35S promoter and the cauliflower terminator was cut with BamHI and the ORF containing fragment was inserted in the unique BamHI site of the binary vector pCGN1589 (21) to result in the plasmid pCISceI.Construction of the target site. Into the EcoRV site of the plasmid pAF100 (22), the Xho I-Acc65I-cut, blunt-ended DNA fragment of the plasmid pAH5 (23) carrying the C-terminal half of an artificial, intron-containing kanamycin resistance gene was cloned....

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Section: Discussionmentioning

confidence: 99%

Two different but related mechanisms are used in plants for the repair of genomic double-strand breaks by homologous recombination.

Puchta

Dujon

Höhn

1996

Proc. Natl. Acad. Sci. U.S.A.

361

283

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“…1 and 3). Because the transient expression of the double-stranded DT-A gene before integration is thought to kill host plant cells (Morton and Hooykaas, 1995), it is assumed that, in the calli that lead to successful GT in our rice system, the entire DT-A gene regions with their promoters in the introduced vector would neither become double stranded nor express transiently before integration Terada, 2004, 2005). Although we do not know whether an intermediate T-DNA molecule for successful GT processes is single stranded or double stranded, we speculate that two DT-A genes placed at both T-DNA ends act effectively to suppress undesirable ectopic events, including OSI, EGT, or simultaneous RI of the positive selection marker and ensure the promotion of an accurate and clean TGT event.…”

Section: Comparison Of Gt Between the Moss And Rice Systemsmentioning

confidence: 99%

Gene Targeting by Homologous Recombination as a Biotechnological Tool for Rice Functional Genomics

et al. 2007

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The modification of an endogenous gene into a designed sequence by homologous recombination, termed gene targeting (GT), has broad implications for basic and applied research. Rice (Oryza sativa), with a sequenced genome of 389 Mb, is one of the most important crops and a model plant for cereals, and the single-copy gene Waxy on chromosome 6 has been modified with a frequency of 1% per surviving callus by GT using a strong positive-negative selection. Because the strategy is independent of gene-specific selection or screening, it is in principle applicable to any gene. However, a gene in the multigene family or a gene carrying repetitive sequences may preclude efficient homologous recombination-promoted GT due to the occurrence of ectopic recombination. Here, we describe an improved GT procedure whereby we obtained nine independent transformed calli having the alcohol dehydrogenase2 (Adh2) gene modified with a frequency of approximately 2% per surviving callus and subsequently isolated eight fertile transgenic plants without the concomitant occurrence of undesirable ectopic events, even though the rice genome carries four Adh genes, including a newly characterized Adh3 gene, and a copy of highly repetitive retroelements is present adjacent to the Adh2 gene. The results indicate that GT using a strong positive-negative selection can be widely applicable to functional genomics in rice and presumably in other higher plants.

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“…The consequences of the introduction of the DT-A gene fused with a strong constitutive promoter as a non-conditional negative selection marker in the somatic recombination pathways for successful gene targeting might be different from those of the introduction of the codA gene, a substrate-dependent negative selection marker. This was because a few toxin molecules produced by transient expression of the DT-A gene on the double-stranded T-DNA intermediate would probably be sufficient to kill the host cell instantly (Czako and An, 1990;Morton and Hooykaas, 1995). The following models are based upon the presumption that in successfully targeted rice cells the two DT-A gene regions flanking the Waxy sequence have not been converted into a double-stranded intermediate after the introduction into the plant nucleus as a singlestranded molecule, because the DT-A gene on the double-stranded T-DNA intermediate can be transiently expressed and a few DT-A molecules produced are sufficient to kill the host cell.…”

Section: Homologous Recombination-dependent Targeting Of An Endogenoumentioning

confidence: 99%

“…After arresting translation, DNA fragmentation associated with cell death was detectable (Day and Irish, 1997). Because the transient expression of the introduced DT-A gene before integration into the genome was thought to kill potential targeted transformants (Morton and Hooykaas, 1995), the substrate-dependent negative selection gene codA was employed as a first choice (Table 2; Thykjaer et al, 1997;Gallego et al, 1999;Wang et al, 2001). As described below, however, only the cell-autonomous and nonconditional negative selection gene DT-A has led to the isolation of successful and reproducible gene targeting by homologous recombination (Terada et al, 2002).…”

Section: Positive-negative Selectionmentioning

confidence: 99%

Modification of Endogenous Natural Genes by Gene Targeting in Rice and Other Higher Plants

Iida

Terada

2005

Plant Mol Biol

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The capability to modify a genomic sequence into a designed sequence is a powerful tool for biologists and breeders to elucidate the function of an individual gene and its cis-acting elements of multigene families in the genome. Gene targeting refers to the alteration of a specific DNA sequence in an endogenous gene at its original locus in the genome. In higher plants, however, the overwhelming occurrence of the random integration of transgenes by non-homologous end-joining is the main obstacle to develop efficient gene targeting. Two approaches have been undertaken to modify a genomic sequence in higher plants- chimeric RNA/DNA oligonucleotide-directed gene targeting to generate a site-specific base conversion, and homologous recombination-dependent gene targeting to produce either a base change or a gene replacement in a sequence-specific manner. The successful and reproducible targeting of an endogenous gene by homologous recombination, independently of gene-specific selection by employing a strong positive-negative selection, has been demonstrated for the first time in rice, an important staple food and a model plant for other cereal species. This review addresses the current status of targeting of an endogenous natural gene in rice and other higher plants and discusses possible models for Agrobacterium- mediated gene targeting by homologous recombination using a strong positive-negative selection.

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Gene replacement

Cited by 27 publications

References 81 publications

Two different but related mechanisms are used in plants for the repair of genomic double-strand breaks by homologous recombination.

Two different but related mechanisms are used in plants for the repair of genomic double-strand breaks by homologous recombination.

Gene Targeting by Homologous Recombination as a Biotechnological Tool for Rice Functional Genomics

Modification of Endogenous Natural Genes by Gene Targeting in Rice and Other Higher Plants

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