Intrachromosomal homologous recombination in whole plants.

A system to assay intrachromosomal homologous recombination during the complete life-cycle of a whole higher eukaryote was set up. Arabidopsis thaliana plants were transformed with a recombination substrate carrying a non-selectable and quantitatively detectable marker gene. The recombination substrates contain two overlapping, non-functional deletion mutants of a chimeric beta-glucuronidase (uidA) gene. Upon recombination, as proven by Southern blot analysis, a functional gene is restored and its product can be detected by histochemical staining. Therefore, cells in which recombination events occurred, and their progeny, can be precisely localized in the whole plant. Recombination was observed in all plant organs examined, from the seed stage until the flowering stage of somatic plant development. Meristematic recombination events revealed cell lineage patterns. Overall recombination frequencies typically were in the range 10(-6)-10(-7) events/genome. Recombination frequencies were found to differ in different organs of particular transgenic lines.     

ZFN‐mediated gene targeting of the Arabidopsis protoporphyrinogen oxidase gene through Agrobacterium‐mediated floral dip transformation

Previously, we showed that ZFN‐mediated induction of double‐strand breaks (DSBs) at the intended recombination site enhanced the frequency of gene targeting (GT) at an artificial target locus using Agrobacterium‐mediated floral dip transformation. Here, we designed zinc finger nucleases (ZFNs) for induction of DSBs in the natural protoporphyrinogen oxidase (PPO) gene, which can be conveniently utilized for GT experiments. Wild‐type Arabidopsis plants and plants expressing the ZFNs were transformed via floral dip transformation with a repair T‐DNA with an incomplete PPO gene, missing the 5′ coding region but containing two mutations rendering the enzyme insensitive to the herbicide butafenacil as well as an extra KpnI site for molecular analysis of GT events. Selection on butafenacil yielded 2 GT events for the wild type with a frequency of 0.8 × 10^?3 per transformation event and 8 GT events for the ZFNs expressing plant line with a frequency of 3.1 × 10^?3 per transformation event. Molecular analysis using PCR and Southern blot analysis showed that 9 of the GT events were so‐called true GT events, repaired via homologous recombination (HR) at the 5′ and the 3′ end of the gene. One plant line contained a PPO gene repaired only at the 5′ end via HR. Most plant lines contained extra randomly integrated T‐DNA copies. Two plant lines did not contain extra T‐DNAs, and the repaired PPO genes in these lines were transmitted to the next generation in a Mendelian fashion.     

Homologous Recombination in Zebrafish ES Cells

Targeted insertion of a plasmid by homologous recombination was demonstrated in zebrafish ES cell cultures. Two selection strategies were used to isolate ES cell colonies that contained targeted plasmid insertions in either the no tail or myostatin I gene. One selection strategy involved the manual isolation of targeted cell colonies that were identified by the loss of fluorescent protein gene expression. A second strategy used the diphtheria toxin A-chain gene in a positive-negative selection approach. Homologous recombination was confirmed by PCR, sequence and Southern blot analysis and colonies isolated using both selection methods were expanded and maintained for multiple passages. The results demonstrate that zebrafish ES cells have potential for use in a cell-mediated gene targeting approach.     

Enhancing gene targeting efficiency in higher plants: rice is on the move

AbstractMeeting the challenge of routine gene targeting (GT) in higher plants is of crucial interest to researchers and plant breeders who are currently in need of a powerful tool to specifically modify a given locus in a genome. Higher plants have long been considered the last lineage resistant to targeting technology. However, a recent report described an efficient method of T-DNA-mediated targeted disruption of a non-selectable locus in rice [Terada et al., Nat Biotechnol 20: 1030–1034 (2002)]. Though this study was an obvious breakthrough, further improvement of GT frequencies may derive from a better understanding of the natural mechanisms that control homologous recombination (HR) processes. In this review, we will focus on what is known about HR and the factors which may hamper the development of routine GT by HR in higher plants. We will also present the current strategies envisaged to overcome these limitations, such as expression of recombination proteins and refinements in the design of the transformation vector.     

Gene targeting in Arabidopsis thaliana.

Summary Gene targeting of a chromosomally integrated transgene in Arabidopsis thaliana is reported. A chimeric gene consisting of the promoter of the 35S RNA of CaMV, the polyadenylation signal of the octopine synthase gene and the coding region of the bacterial hygromycin phosphotransferase gene (hpt), which was rendered non-functional by deletion of 19 bp, was introduced into the genome of A. thaliana using Agrobacterium-mediated gene transfer. A total of 3.46 x 10⁸ protoplasts isolated from 17 independent transgenic Arabidopsis lines harbouring the defective chimeric hpt gene were transformed via direct gene transfer using various DNA forms containing only the intact coding region of the hpt gene. Out of 150 hygromycin-resistant colonies appearing in the course of these experiments, four were the result of targeted recombination of the incoming DNA with the defective chromosomal locus as revealed by PCR and Southern blot analysis. Comparison with the number of transformants obtained when an hpt gene controlled by a promoter and terminator from the nopaline synthase gene was employed results in a maximal ratio of homologous to non-homologous transformation in A. thaliana of 1 x 10^–4.     

Efficient removal of loxP-flanked DNA sequences in a gene-targeted locus by transient expression of Cre recombinase in fertilized eggs

The bacteriophage P1 Cre/loxP site-specific recombination system is a useful tool for engineering chromosomal changes in animal cells. Transient expression of the Cre recombinase gene directly introduced into fertilized eggs by pronuclear injection has been reported to provide an efficient method of transgene modulation in fertilized eggs. In the present study, we examined the efficacy of this method to remove loxP-flanked DNA sequences in a gene-targeted locus in fertilized eggs. We replaced a part of the T-cell receptor γ (TCR Vγ) locus with homologous sequences containing a loxP-flanked neogene in mouse embryonic stem (ES) cells by gene-targeting technique. The resulting ES cell clones containing the mutant allele (Vγ^LNL) were used to generate chimeric mice by blastocyst injection. Eight male chimeras were bred with superovulated wild-type female mice. One hundred and seventy-six fertilized eggs were collected, and subjected to pronuclear injection of the Cre expression plasmid, pCAGGS-Cre, of a covalently closed circular form. Three out of 11 pups inherited the targeted Vγ locus. The inherited targeted allele of these 3 mice was shown to have undergone Cre-mediated recombination, resulting in a deletion of the loxP-flanked sequences (Vγ^Δ) as shown by Southern blot analysis of DNA from tail biopsies. All 3 founder mutant mice were capable of transmitting the Vγ^Δ locus to their offspring. The other 8 pups carried only wild-type alleles. There were no pups carrying the unrecombined Vγ^LNL locus. Thus, the frequency of Cre-mediated recombination was 100% (3/3) with this method. In contrast, when closed circular pCAGGS-Cre plasmid was introduced into ES cells by electroporation, the recombination frequency of the Vγ^LNL locus was 9.6%. These results indicated that our system based on transient expression of the Cre recombinase gene directly introduced into fertilized eggs by pronuclear injection provides a fast and efficient method for generating mutant mice with desired deletions or translocations in target genes. Mol Reprod Dev 46:109–113, 1997. © 1997 Wiley-Liss, Inc.     

Frequency and character of alternative somatic recombination fates of paralogous genes during T-DNA integration

A synthetic RBCSB gene cluster was transformed into Arabidopsis in order to simultaneously evaluate the frequency and character of somatic illegitimate recombination, homologous recombination, and targeted gene replacement events associated with T-DNA-mediated transformation. The most frequent type of recombination event observed was illegitimate integration of the T-DNA without activation of the silent ΔRBCS1B: LUC transgene. Sixteen luc⁺ (firefly luciferase positive) T1 plants were isolated. Six of these were due to illegitimate recombination events resulting in a gene trapping effect. Nine resulted from homologous recombination between paralogous RBCSB sequences associated with T-DNA integration. The frequency of somatic homologous recombination associated with T-DNA integration was almost 200 times higher than previously reported rates of meiotic homologous recombination with the same genes. The distribution of (somatic homologous) recombination resolution sites generally fits a fractional interval length model. However, a small region adjacent to an indel showed a significant over-representation of resolution sites, suggesting that DNA mismatch recognition may also play an important role in the positioning of somatic resolution sites. The frequency of somatic resolution within exon-2 was significantly different from that previously observed during meiotic recombination. Electronic Supplementary Material Supplementary material is available for this article at     

Efficient transformation of Arabidopsis thaliana using direct gene transfer to protoplasts

Summary Direct gene transfer has proved to be an efficient transformation method for arabidopsis thaliana, a member of the Brassicaceae. Transgenic Arabidopsis plants resistant to hygromycin B have been regenerated from mesophyll protoplasts treated with polyethylene glycol and plasmid DNA carrying the hygromycin phosphotransferase (HPT) gene under the control of the 35 S promoter of cauliflower mosaic virus. The transformation procedure reproducibly yields transformants at frequencies of approximately 1×10^-4 (based on the number of protoplasts treated) or 5% (based on the number of regenerating calli). DNA from plants regenerated from hygromycin resistant colonies was analysed by Southern blot hybridization demonstrating that the foreign gene is stably integrated into the plant chromosome. Genetic analysis of several hygromycin resistant plants showed that the HPT gene is transmitted to the progeny. Transformation experiments performed with a selectable and a non-selectable gene on separate plasmids resulted in a co-transformation rate of functionally active copies in about 25% of the transformants analysed. Hence this approach can be used to introduce non-selectable genes into the Arabidopsis genome.     

Genetic characterization of the Escherichia coli cyclopropane fatty acid (cfa) locus and neighboring loci

Summary The phenotypically silent cyclopropane fatty acid synthesis (cfa) gene of Escherichia coli K-12 has been located on the genetic linkage map. This was accomplished by integrating (via homologous recombination) the selectable marker of a recombinant plasmid into the host chromosome near the cfa locus. This integration allowed the subsequent isolation of a cfa-linked transposon Tn10 insertion. Genetic mapping of the Tn10 insertion, using conventional techniques, placed the cfa locus at min 36.5 on the linkage map in the vicinity of several other non-selectable markers. We ordered cfa and these other loci by three-factor transductional analyses. Selection for excision of the Tn10 element resulted in several types of mutants which harbor mutations of cfa and of neighboring genes, presumably as a consequence of Tn10-catalyzed chromosomal rearrangements.     

Integration of Double-Fluorescence Expression Vectors into Zebrafish Genome for the Selection of Site-Directed Knockout/Knockin

Production of zebrafish by modifying endogenous growth hormone (GH) gene through homologous recombination is described here. We first constructed the targeting vectors pGHT1.7k and pGHT2.8k, which were used for the knockout/knockin of the endogenous GH gene of zebrafish, and injected these two vectors into the embryos of zebrafish. Overall, the rate of targeted integration with the characteristic of germ line transmission in zebrafish was 1.7×10⁻⁶. In one experimental patch, the integrating efficiency of pGHT2.8k was higher than that of pGHT1.7k, but the lethal effect of pGHT2.8k was stronger than that of pGHT1.7k. The clones with the correct integration of target genes were identified by a simple screening procedure based on green fluorescent protein (GFP) and RFP dual selection, which corresponded to homologous recombination and random insertion, respectively. The potential homologous recombination zebrafish was further bred to produce a heterozygous F₁ generation, selected based on the presence of GFP. The potential targeted integration of exogenous GH genes into a zebrafish genome at the P₀ generation was further verified by polymerase chain reaction and Southern blot analysis. Approximately 2.5% of potential founder knockout and knockin zebrafish had the characteristic of germ line transmission. In this study, we developed an efficient method for producing the targeted gene modification in zebrafish for future studies on genetic modifications and gene functions using this model organism. ^★Equal contributions to this article.     

Precision genome editing in plants via gene targeting and piggyBac‐mediated marker excision

Precise genome engineering via homologous recombination (HR)‐mediated gene targeting (GT) has become an essential tool in molecular breeding as well as in basic plant science. As HR‐mediated GT is an extremely rare event, positive–negative selection has been used extensively in flowering plants to isolate cells in which GT has occurred. In order to utilize GT as a methodology for precision mutagenesis, the positive selectable marker gene should be completely eliminated from the GT locus. Here, we introduce targeted point mutations conferring resistance to herbicide into the rice acetolactate synthase (ALS) gene via GT with subsequent marker excision by piggyBac transposition. Almost all regenerated plants expressing piggyBac transposase contained exclusively targeted point mutations without concomitant re‐integration of the transposon, resulting in these progeny showing a herbicide bispyribac sodium (BS)‐tolerant phenotype. This approach was also applied successfully to the editing of a microRNA targeting site in the rice cleistogamy 1 gene. Therefore, our approach provides a general strategy for the targeted modification of endogenous genes in plants.     

Large scale extraction of high quality moss DNA

For several years Physcomitrella patens Hedw. was the only land plant allowing targeted gene knockout via homologous recombination, which provides an efficient and elegant tool for the analysis of gene functions. The moss Ceratodon purpureus Hedw. was recently shown to have similar capabilities. However, extraction of high quality total DNA from both moss species — necessary for unambiguous proof of successful gene targeting events — is still a severe problem. Here, we report an improved DNA isolation protocol for moss filaments, which is fast, reliable, cheap, quick, and easy. It results in satisfying yields of DNA suitable for PCR and Southern blotting. The modified extraction procedure was additionally tested successfully for the alga Mougeotia scalaris Hass. as well as the higher plant Arabidopsis thaliana L. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 4, pp. 634–638. The text was submitted by the authors in English.     

Molecular cloning of alpha-amylase genes fromDrosophila melanogaster. III. An inversion at theAmy locus in an amylase-null strain

Overlapping clones of the structural gene region for alpha-amylase,Amy, were isolated from a lambda EMBL4 library containing genomic DNA fragments from an amylase-null strain ofDrosophila melanogaster. Southern blot analysis and restriction endonuclease mapping of the cloned region indicate that it contains anAmy gene duplication within an inverted repeat sequence as is characteristic of the genomic arrangement for this species. Spacing between the cloned gene copies is similar to that commonly found in other strains. Evidence is presented for the presence of an inversion 4 to 9 kb in length within the clonedAmy region of the null strain. We postulate a causal relationship between the presence of the inversion and the failure of individuals from the null strain to express amylase. A model is proposed that suggests the inversion may have arisen through intramolecular (or sister-strand) recombination mediated by homologous pairing of the inverted repeat sequences at theAmy locus.This research was supported by NIH Grant GM25255.     

Double-strand gap repair in a mammalian gene targeting reaction.

To better understand the mechanism of homologous recombination in mammalian cells that facilitates gene targeting, we have analyzed the recombination reaction that inserts a plasmid into a homologous chromosomal locus in mouse embryonic stem cells. A partially deleted HPRT gene was targeted with various plasmids capable of correcting the mutation at this locus, and HPRT+ recombinants were directly selected in HAT medium. The structures of the recombinant loci were then determined by genomic Southern blot hybridizations. We demonstrate that plasmid gaps of 200, 600, and 2,500 bp are efficiently repaired during the integrative recombination reaction. Targeting plasmids that carry a double-strand break or gap in the region of DNA homologous to the target locus produce 33- to 140-fold more hypoxanthine-aminopterin-thymidine-resistant recombinants than did these same plasmids introduced in their uncut (supercoiled) forms. Our data suggest that double-strand gaps and breaks may be enlarged prior to the repair reaction since sequence heterologies carried by the incoming plasmids located close to them are often lost. These results extend the known similarities between mammalian and yeast recombination mechanisms and suggest several features of the insertional (O-type) gene targeting reaction that should be considered when one is designing mammalian gene targeting experiments.     

Gene targeting in maize by somatic ectopic recombination

Low transformation efficiency and high background of non‐targeted events are major constraints to gene targeting in plants. We demonstrate here applicability in maize of a system that reduces the constraint from transformation efficiency. The system requires regenerable transformants in which all of the following elements are stably integrated in the genome: (i) donor DNA with the gene of interest adjacent to sequence for repair of a defective selectable marker, (ii) sequence encoding a rare‐cutting endonuclease such as I‐SceI, (iii) a target locus (TL) comprising the defective selectable marker and I‐SceI cleavage site. Typically, this requires additional markers for the integration of the donor and target sequences, which may be assembled through cross‐pollination of separate transformants. Inducible expression of I‐SceI then cleaves the TL and facilitates homologous recombination, which is assayed by selection for the repaired marker. We used bar and gfp markers to identify assembled transformants, a dexamethasone‐inducible I‐SceI::GR protein, and selection for recombination events that restored an intact nptII. Applying this strategy to callus permitted the selection of recombination into the TL at a frequency of 0.085% per extracted immature embryo (29% of recombinants). Our results also indicate that excision of the donor locus (DL) through the use of flanking I‐SceI cleavage sites may be unnecessary, and a source of unwanted repair events at the DL. The system allows production, from each assembled transformant, of many cells that subsequently can be treated to induce gene targeting. This may facilitate gene targeting in plant species for which transformation efficiencies are otherwise limiting.     

Improved techniques for endogenous epitope tagging and gene deletion in Toxoplasma gondii

Toxoplasma gondii is an excellent model organism for studies on the biology of the Apicomplexa due to its ease of in vitro cultivation and genetic manipulation. Large-scale reverse genetic studies in T. gondii have, however, been difficult due to the low frequency of homologous recombination. Efforts to ensure homologous recombination have necessitated engineering long flanking regions in the targeting construct. This requirement makes it difficult to engineer chromosomally targeted epitope tags or gene knock out constructs only by restriction enzyme mediated cloning steps. To address this issue we employed multisite Gateway® recombination techniques to generate chromosomal gene manipulation targeting constructs. Incorporation of 1.5 to 2.0 kb flanking homologous sequences in PCR generated targeting constructs resulted in 90% homologous recombination events in wild type T. gondii (RH strain) as determined by epitope tagging and target gene deletion experiments. Furthermore, we report that split marker constructs were equally efficient for targeted gene disruptions using the T. gondii UPRT gene locus as a test case. The methods described in this paper represent an improved strategy for efficient epitope tagging and gene disruptions in T. gondii.     

Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers

Novel ‘super-binary’ vectors that carried two separate T-DNAs were constructed. One T-DNA contained a drug-resistance, selection-marker gene and the other contained a gene for β-glucuronidase (GUS). A large number of tobacco (Nicotiana tabacum L.) and rice (Oryza sativa L.) transformants were produced by Agrobacterium tume-faciens LBA4404 that carried the vectors. Frequency of co-transformation with the two T-DNAs was greater than 47%. GUS-positive, drug-sensitive progeny were obtained from more than half of the co-transformants. Molecular analyses by Southern hybridization and polymerase chain reactions confirmed integration and segregation of the T-DNAs. Thus, the non-selectable T-DNA that was genetically separable from the selection marker was integrated into more than a quarter of the initial, drug-resistant transformants. Since various DNA fragments may be inserted into the non-selectable T-DNA by a simple procedure, these vectors will likely be very useful for the production of marker-free transformants of diverse plant species. Delivery of two T-DNAs to plants from mixtures of A. tumefaciens was also tested, but frequency of co-transformation was relatively low.     

Efficient gene targeting in the filamentous fungusAlternaria alternata

To characterize homologous recombination of transforming DNA in the filamentous fungusAlternaria alternata, we have compared the frequencies of gene targeting by circular and linear DNA fragments in the fungus. TheA. alternata BRM1 gene, which is an essential gene for melanin biosynthesis, was selected as a target locus.BRM1 targeting events are easily identified because loss of function leads to a change in mycelial color from black to light brown. We constructed targeting vectors by inserting 0.6 to 3.1 kb internalBRM1 segments into a plasmid containing the hygromycin B phosphotransferase gene. When circular plasmids were used, melanin-deficient (Me1^–) transformants accounted for 30 to 80% of hygromycin B-resistant (Hy^R) transformants, correlating closely with the size of theBRM1 segment in the transforming DNA. Restriction enzyme digestion within theBRM1 region greatly enhanced the frequency of gene targeting: integration of the linear plasmids was almost completely attributable to homologous recombination, regardless of the size of theBRM1 segments. Plasmids carrying bothBRM1 segments and rDNA segments were transformed into the fungus to examine the effect of the number of target copies on homologous recombination. Using the circular plasmids, Me1^– transformants accounted for only 5% of Hy^R transformants. In contrast, when the linear plasmid produced by restriction enzyme digestion within theBRM1 segment was used, almost all transformants were Me1^–. These results indicate that homologous integration of circular molecules inA. alternata is sensitive to the length of homology and the number of targets, and that double-strand breaks in transforming DNA greatly enhance homologous recombination.