The EMBO Journal vol.7 no.13 pp.4021 -4026, 1988 Gene targeting in plants Jerzy Paszkowski, Markus Baur, Although integration of transforming DNA into the Augustyn Bogucki and Ingo Potrykus homologous chromosomal DNA occurs efficiently in yeast, other fungi and Dictyostelium discoideum (Hinnen et al., Friedrich Miescher Institut, PO Box 2543, CH4002 Basel, 1978; De Lozanne et al., 1987; Miller et al., 1987), Switzerland non-homologous ('illegitimate') integration of foreign DNA Present address: Swiss Federal Institute of Technology, Institute of into the genome of higher eukaryotic cells makes it difficult Plant Sciences, ETH-Zentrum, Universitatstrasse 2, CH-8092 Zurich, to assay the frequency of gene targeting to a desired locus. Switzerland Only recently has the application of selection (Lin et al., Communicated by J.-D.Rochaix 1985; Thomas et al., 1986; Doetschman et al., 1987; Song et al., 1987; Thomas and Capecchi, 1987) and screening Although the generation of transgenic plants is now (Smithies et al., 1985) systems allowed comparison of the routine, the integration of foreign genetic information has frequencies of homologous and illegitimate integration of so far been at random sites in the genome. We now transformed DNA in cultured mammalian cells. In plants present evidence for directed integration into a predicted the high transformation frequencies reported for DNA location in the host plant genome. Protoplasts of molecules with no homology to the plant genome (Shillito transgenic tobacco (Nicotiana tabacum) plants carrying et al., 1985; Negrutiu et al., 1987) indicate efficient copies of a partial, non-functional drug-resistance gene illegitimate integration. Therefore, the detection of gene in the nuclear DNA were used as recipients for DNA targeting through homologous DNA recombination requires molecules containing the missing part of the gene. the utilization of a rigorous selection system such as selection Molecular and genetic data confirm the integration of for kanamycin resistance conferred by a gene coding for the foreign DNA through homologous recombination neomycin phosphotransferase [APH(3')II] which has within overlapping parts of the protein coding region, provided tight selection both at the cellular and the plant level resulting in the formation of an active gene in the host (Herrera-Estrella et al., 1983; Paszkowski et al., 1984). chromosome. This approach is referred to as gene We have constructed five strains of transgenic tobacco targeting. The gene targeting frequency (the number of plants containing chromosomal copies of a non-functional, drug-resistant clones resulting from gene correction partially deleted, hybrid A.PH(3')II gene. Protoplasts of these compared to the number of resistant clones from parallel plants were used for DNA mediated direct transformation experiments with a similar non-interrupted hybrid gene) with the missing part of the gene in order to correct its was 0.5-4.2 x 1O-4. These experiments demonstrate genomic counterpart. Here we report results of these the possibility of producing transgenic plants with desired experiments resulting in the desired modification of the modifications to a specific nuclear gene. genomic target gene sequence. Key words: gene targeting/homologous DNA recombination/ protoplast/transformation/transgenic tobacco plants Results Experimental design and the construction of the target plant strains Introduction The bacterial APH(3')Il gene under the control of plant viral There have been important advances in the past few years expression signals (Figure IA and B) was used in the in gene transfer techniques with plants. The most common construction of two sets of complementing plasmid molecules technique uses Agrobacterium tumefaciens as a vector which could form an active gene through homologous (Rogers et al., 1986). In addition, direct gene transfer to recombination (Figure IA). One set (3' deletions) contained protoplasts (Paszkowski et al., 1984; Hain et al., 1985; the promoter region and 5' part of the structural gene (Figure Shillito et al., 1985; Negrutiu et al., 1987), protoplast fusion IA;b,f). The other set (5' deletions) contained the 3' with liposomes containing DNA (Deshayes et al., 1985), end of the APH(3')II structural gene and the signals for intranuclear microinjection of protoplasts (Crossway et polyadenylation (Figure IA;c,d,g). With combinations of al., 1986; Reich et al., 1986) and macroinjection into these two types of plasmids we were able to choose defined meristematic inflorescences (De la Pena et al., 1987) are lengths of homology in the central part of the APH(3')II available. gene. Details of the plasmid constructions are described in Whatever the method, incoming DNA integrates the legend to Figure 1. efficiently into the nuclear genome but at random locations A series of transgenic tobacco lines were produced as (Potrykus et al., 1985; Ambros et al., 1986; Nagy et al., recipients for gene targeting by introducing the 5' or 3' 1986; Wallroth et al., 1986). It would be useful to be able deletion derivatives of the APH(3')II gene (Figure 1A;b,c,d) to modify plant genes in situ at their natural position in the into mesophyll protoplasts using co-transformation (Schocher genome or to deliver foreign DNA into a predicted genomic et al., 1986) with a selectable gene for hygromycin resistance location. (Figure 1B). Seven random hygromycin-resistant clones for (©)IRL Press Limited, Oxford, England 4021 J.Paszkowski et al. each deletion derivative were screened by Southern blot the constructs into the nuclear genome was verified by hybridization. Five clones were chosen (NI, R2, R3, JI, their mitotic stability in culture and transmission through J2) containing the expected gene structure for particular male gametes (tested by Southern blot analysis on progeny APH(3')II deletion derivatives of plasmid pABDI (Figure from crosses to wild-type). Ro hemizygous plants were lA;b,c,d). The copy numbers of the truncated APH(3')II propagated as shoot cultures as a source of protoplasts. gene (intact deletion derivatives) in the cell lines J 1, J2, N1, R2 and R3 were estimated by Southern blot reconstruction Gene targeting to be - 3, 7, 5, 2 and 10, respectively. Previous experiments All five lines were used in targeting experiments (Table with direct gene transfer into tobacco protoplasts have shown I). Plasmid DNA containing complementing parts of the that although high copy numbers of integrated DNA are often APH(3')ll hybrid gene was introduced by direct gene transfer found, in 77 % of cases the integrated DNA segregates as into protoplasts of the corresponding plant strains as follows: one locus (Potrykus et al., 1987). Thus the copy numbers plasmid pHP28ANarI (Figure lA;g) into strain NI (Figure given above probably represent DNA integrated in one locus lA;b), plasmid pHP28zASphl (Figure lA;f) into strains R2, in the plant genome and these copies can be often found as R3, JI and J2 (Figure lA;c,d). From three independent concatamers (Czernilofsky et al., 1986). experiments involving a total of 1.68 x 108 protoplasts Transgenic plants were regenerated and the integration of (Table I), eight kanamycin-resistant cell clones were obtained A 123 pABDI (5265b.p.) NdeI -137 0 176 205 52%i 805 1007 HiIEcoRVHZindI Pstl Ball Sphl HindfI EcoRV Smali ar Ps I r- r-t\\ \ < Bo( I b ANi 'LI, Norl ri c -11 rj-- R2,R3 H indI Pst l IS d i I f- -J1,32 -240 0 805 1007 EcoRV Hind m HindM EcoRV I.. 1 r- -r-r e r_IiuI fIssPr"/////////////////vNE~~ULEZI2 Sph I 9 Norl h = h arl coRI -24Q 0 123 176 205 528 805 EcoRI Sphl Ndel E -RVHindM Narl Pstl Boil SphI Hindm < ~~~~~~pHP28 (4715 hp.) B EcoRVL Hind m Eco RV APH(3')U gene from pABDI EcoR _I EcoR --b. --c ~ ~ H P23 pLG88 -pA BDHB 4022 Gene targeting in plants from recipient strains J2 and R3. A positive APH(3')II between the deletions should result in a marker gene with activity test of the clones (data not shown) confirmed the a new combination of flanking segments, distinguishable nature of this resistance in each case. No kanamycin-resistant from the two constructs previously existing (pABDI and clones were observed in corresponding control experiments pHP28). Southern analysis of four J2 complemented clones where wild-type SRI protoplasts were used or where non- revealed a predicted EcoRV fragment of the new marker complementary deletion derivatives were supplied to proto- gene (Figure 2A; lanes 3-6). This confirms recombination plasts of the five lines (a total of 1.1 x 10 protoplasts). in the central region of homology of the two deletion The frequency of the appearance of kanamycin-resistant derivatives, and rules out the possibility of transformation clones per treated protoplast was compared to the frequency with contaminating parental plasmids. A reprobing of the of transformation with a similar, non-interrupted hybrid gene filters with probes specific for 5' part and 3' part of the gene (plasmid pHP28) in parallel treatments. The ratio (the relative [for 5' fragment EcoRV(-240)-PstI(176) and Hin- targeting frequency, RTF) varied from 5.4 x 10-5 for line dII(O)-PstI(176) and for 3' PstI(176)-HindllI(805), R3 to 0.83-4.2 x 10-4 (reproducible gene targeting) in Figure 1] revealed that both probes hybridized with the same line J2 (Table I). The expression signals of the gene predicted EcoRV fragment as the complete probe. Southern analysis to result from targeting are identical to those of the intact also showed that not all copies of the integrated APH(3')II reference gene. The only difference between these two genes deletions were corrected: (as seen in Figure 2B, some 634 is that the poly(A) addition site in the reference gene is bp HindlIl fragments were not converted to new 805 bp 280 bp closer to the coding sequence.