Design of a novel system for the construction of vectors for Agrobacterium-mediated plant transformation

Summary The loxP-Cre site-specific recombination system of phage P1 was used to develop a novel strategy to construct cointegrate vectors for Agrobacterium-mediated plant transformation. A pTi disarmed helper plasmid (pAL1166) was constructed by replacing the oncogenic T-DNA by a loxP sequence and a spectinomycin resistance marker in the octopine-type pTiB6 plasmid. The cre gene was cloned into an unstable incP plasmid. A third plasmid, which did not replicate in Agrobacterium and contained another loxP sequence together with a kanamycin resistance marker, was used to test the system. Electroporation of this third plasmid into an Agrobacterium strain harbouring both pAL1166 and the Cre-encoding plasmid resulted in kanamycin-resistant cells containing a cointegrate between pAL1166 and the incoming plasmid. Cointegration occurred by Cre-mediated recombination at the loxP sites, and the cointegrate was stabilized in the Agrobacterium cells by the loss of the Cre-encoding plasmid shortly after the recombination event had taken place.     

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.     

pBECKS2000: a novel plasmid series for the facile creation of complex binary vectors, which incorporates "clean-gene" facilities

A new plasmid series has been created for Agrobacterium-mediated plant transformation. The pBECKS2000 series of binary vectors exploits the Cre/loxP site-specific recombinase system to facilitate the construction of complex T-DNA vectors. The new plasmids enable the rapid generation of T-DNA vectors in which multiple genes are linked, without relying on the availability of purpose-built cassette systems or demanding complex, and therefore inefficient, ligation reactions. The vectors incorporate facilities for the removal of transformation markers from transgenic plants, while still permitting simple in vitro manipulations of the T-DNA vectors. A `shuttle' or intermediate plasmid approach has been employed. This permits independent ligation strategies to be used for two gene sets. The intermediate plasmid sequence is incorporated into the binary vector through a plasmid co-integration reaction which is mediated by the Cre/loxP site-specific recombinase system. This reaction is carried out within Agrobacterium cells. Recombinant clones, carrying the co-integrative binary plasmid form, are selected directly using the antibiotic resistance marker carried on the intermediate plasmid. This strategy facilitates production of co-integrative T-DNA binary vector forms which are appropriate for either (1) transfer to and integration within the plant genome of target and marker genes as a single T-DNA unit; (2) transfer and integration of target and marker genes as a single T-DNA unit but with a Cre/loxP facility for site-specific excision of marker genes from the plant genome; or (3) co-transfer of target and marker genes as two independent T-DNAs within a single-strain Agrobacterium system, providing the potential for segregational loss of marker genes. Received: 30 July 1998 / Accepted: 2 November 1998     

Cre-mediated germline mosaicism: a method allowing rapid generation of several alleles of a target gene

Conditional gene targeting uses the insertion of expression cassettes for the selection of targeted embryonic stem cells. The presence of these cassettes in the final targeted chromosomal locus may affect the normal expression of the targeted gene and produce interesting knock down phenotypes. We show here that the selection cassette may then be selectively removed in vivo, using three appropriately positioned loxP sites in the targeted gene and the transgenic mouse EIIaCre. This strategy was applied to two different target genes and we demonstrated that it is reliable and reproducible. First, we generated double transgenic EIIaCre/loxP mice (F1) that showed variable degrees of mosaicism for partially Cre-recombined floxed alleles. Efficiency of EIIaCre at creating mosaicism was dependent on the target gene and on parental transmission of the transgene. The segregation of partially recombined alleles and EIIaCre transgene was obtained in the next generation using mosaic F1 males. Mosaic females were unsuitable for this purpose because they systematically generated complete excisions during oogenesis. Our strategy is applicable to other approaches based on three loxP sites. As this procedure allows generation of knock down (presence of neo), knockout (total exision of the loxP-flanked sequences) and floxed substrains (excision of the selection cassette) from a single, targeted germline mutation and in a single experiment, its use may become more widespread in conditional mutagenesis.     

Efficient DNA cassette exchange in mouse embryonic stem cells by staggered positive-negative selection

Recombinase-mediated cassette exchange (RMCE), when applied to mouse embryonic stem (ES) cells, promises to increase the ease with which genetic alterations can be introduced into targeted genomic loci in the mouse. However, existing selection strategies for identifying ES cells in which replacement DNA cassettes from a carrier plasmid have been exchanged correctly into a defined locus are suboptimal. Here, we report the generation in mouse ES cells of a loxed cassette acceptor (LCA) allele within the glucokinase (gk) gene locus. Using the gkLCA as a test allele, we developed a staggered positive-negative selection strategy that facilitates efficient identification of ES cell clones in which a DNA replacement cassette from a carrier plasmid has been exchanged correctly into the gkLCA allele. This selection strategy, by facilitating more efficient production of ES cell clones with various replacement DNA cassettes, should accelerate targeted repetitive introduction of gene modifications into the mouse.     

An inbred 129SvEv GFPCre transgenic mouse that deletes loxP-flanked genes in all tissues

A common method for generating mice with subtle genetic manipulations uses homologous recombination (HR) in embryonic stem (ES) cells to replace a wild-type gene with a slightly modified one. Generally, a drug resistance gene is inserted with the modified gene to select correctly targeted clones. Often, however, the presence of this drug resistance gene interferes with the normal locus and creates a null or hypomorphic allele. Flanking of the selectable marker by loxP sites followed by Cre-mediated deletion after drug selection can overcome this problem. The simplest method used to remove a loxP-flanked selectable marker is to breed an animal carrying a loxP-flanked drug resistance gene to an animal that expresses Cre recombinase in the germline. To date only outbred transgenic mice are available for this purpose. This can be problematic for phenotypic analysis in many organ systems, including the brain, and for the analysis of behavior. While attempting to make 129S6/SvEvTac inbred background (isogenic to our ES cells) mice that express Cre under the control of several tissue-specific promoters, we serendipitously generated a line that excises loxP-flanked drug resistance genes in all tissues, including the germline. This reagent allows deletion of loxP-flanked sequences while maintaining the mutation on an inbred background.     

A Cre/loxP-deleter transgenic line in mouse strain 129S1/SvImJ

A Cre recombinase expression cassette was inserted into the X-linked Hprt locus by gene targeting in a mouse embryonic stem (ES) cell line isogenic to strain 129S1/SvImJ (129S1), then the transgene was introduced into 129S1 mice through ES cell chimeras. When females hemizygous for this transgene were mated to males carrying a neomycin selection cassette flanked by loxP sites, the cassette was always excised regardless of Cre inheritance and without detectable mosaicism. The usefulness of this "Cre-deleter" transgenic line is in its efficiency and defined genetic status in terms of mouse strain and location of the transgene.     

pBECKS2000: a novel plasmid series for the facile creation of complex binary vectors, which incorporates “clean-gene” facilities

A new plasmid series has been created for Agrobacterium-mediated plant transformation. The pBECKS2000 series of binary vectors exploits the Cre/loxP site-specific recombinase system to facilitate the construction of complex T-DNA vectors. The new plasmids enable the rapid generation of T-DNA vectors in which multiple genes are linked, without relying on the availability of purpose-built cassette systems or demanding complex, and therefore inefficient, ligation reactions. The vectors incorporate facilities for the removal of transformation markers from transgenic plants, while still permitting simple in vitro manipulations of the T-DNA vectors. A `shuttle' or intermediate plasmid approach has been employed. This permits independent ligation strategies to be used for two gene sets. The intermediate plasmid sequence is incorporated into the binary vector through a plasmid co-integration reaction which is mediated by the Cre/loxP site-specific recombinase system. This reaction is carried out within Agrobacterium cells. Recombinant clones, carrying the co-integrative binary plasmid form, are selected directly using the antibiotic resistance marker carried on the intermediate plasmid. This strategy facilitates production of co-integrative T-DNA binary vector forms which are appropriate for either (1) transfer to and integration within the plant genome of target and marker genes as a single T-DNA unit; (2) transfer and integration of target and marker genes as a single T-DNA unit but with a Cre/loxP facility for site-specific excision of marker genes from the plant genome; or (3) co-transfer of target and marker genes as two independent T-DNAs within a single-strain Agrobacterium system, providing the potential for segregational loss of marker genes.     

Targeted transgene integration in plant cells using designed zinc finger nucleases

Targeted transgene integration in plants remains a significant technical challenge for both basic and applied research. Here it is reported that designed zinc finger nucleases (ZFNs) can drive site-directed DNA integration into transgenic and native gene loci. A dimer of designed 4-finger ZFNs enabled intra-chromosomal reconstitution of a disabled gfp reporter gene and site-specific transgene integration into chromosomal reporter loci following co-transformation of tobacco cell cultures with a donor construct comprised of sequences necessary to complement a non-functional pat herbicide resistance gene. In addition, a yeast-based assay was used to identify ZFNs capable of cleaving a native endochitinase gene. Agrobacterium delivery of a Ti plasmid harboring both the ZFNs and a donor DNA construct comprising a pat herbicide resistance gene cassette flanked by short stretches of homology to the endochitinase locus yielded up to 10% targeted, homology-directed transgene integration precisely into the ZFN cleavage site. Given that ZFNs can be designed to recognize a wide range of target sequences, these data point toward a novel approach for targeted gene addition, replacement and trait stacking in plants.     

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.     

A new site-specific recombinase-mediated system for targeted multiple genomic deletions employing chimeric loxP and mrpS sites

A newly designed site-specific recombination system is presented which allows multiple targeted markerless deletions. The most frequently used tool for removing selection markers or to introduce genes by recombination-mediated cassette exchange is the Cre/loxP system. Many mutant loxP sites have been created for this purpose. However, this study presents a chimeric mutant loxP site denoted mroxP-site. The mroxP site consists of one Cre (loxP/2) and one MrpA (mrpS/2) binding site separated by a palindromic 6-bp spacer sequence. Two mroxP-sites can be recombined by Cre recombinase in head-to-tail as well as in head-to-head orientation. In the head-to-head orientation and the loxP half-sites inside, Cre removes the loxP half-sites during site-specific recombination, creating a new site, mrmrP. The new site is essentially a mrpS site with a palindromic spacer and cannot be used by Cre for recombination anymore. It does, however, present a substrate for the recombinase MrpA. This new system has been successfully applied introducing multiple targeted gene deletions into the Escherichia coli genome. Similar to Cre/loxP and FLP/FRT, this system may be adapted for genetic engineering of other pro- and eukaryotes.     

Site-specific integration of transgene targeting an endogenous <Emphasis Type="Italic">lox-</Emphasis>like site in early mouse embryos

Functional lox-like sequences have been identified within the yeast and mammalian genome. These hetero-specific lox sites also allow Cre recombinase to specifically target efficient integration of exogenous DNA into the endogenous pseudo-lox (ψlox) sequences that occur naturally in the host genome using a Cre/loxP integrative recombination system. We investigated whether the Cre/ψlox system is useful for site-specific integration of transgenes and for improving the production efficiency of transgenic animals. This is the first report on Cre-mediated integrative recombination targeting an endogenous lox-like sequence termed pseudo-loxm5 (ψloxm5) in early mouse embryos. We characterized the Cre/ψloxm5 system in embryonic environment. Cre-expressing plasmid and a transgene (CMV/LacZ gene) flanked by ψloxm5 and ψloxcorem5 sites were co-microinjected into the pronucleus of fertilized mouse oocytes. The injected eggs were transferred into foster mothers, and the recombination products were investigated. The results show that the ψloxm5 site is an active substrate for Cre-mediated recombination in the mouse embryonic environment. The transgenesis efficiency was up to 27% (6/22). The site-specific integration of the transgene into the endogenous ψloxm5 site was found in 50 % of the transgenic pups. Our findings demonstrated that the Cre/ψloxm5 integrative recombination system is an efficient and simple strategy for targeting an endogenous lox-like site in mammalian embryos.     

Enhanced expression of human cDNA by phosphoglycerate kinase promoter-puromycin cassette in the mouse transthyretin locus

To produce a humanized mouse, it is critical to obtain a correct expression of a human gene/cDNA after insertion into a mouse locus. We previously generated a targeted allele in which the PGK-neo cassette, flanked by lox71 and loxP, was inserted into the first exon of the mouse endogenous transthyretin (Ttr) gene in ES cells. Using these ES cells, we showed that a human transthyretin (TTR) cDNA with the PGK-puro cassette can be efficiently inserted into this locus by Cre-mediated recombination, and that the human TTR cDNA was expressed in a tissue-specific manner under the control of the mouse endogenous Ttr promoter. To examine whether the PGK-puro cassette or IRES could affect the expression of human TTR cDNA, we generated four mouse lines using Cre and Flp-mediated recombination. The mouse line containing the PGK-puro cassette, but not IRES, exhibited quantitatively and temporally similar expression of human TTR cDNA. Removal of the PGK-puro cassette significantly downregulated the expression of the cDNA. The insertion of IRES sequence upstream of the human TTR cDNA resulted in decreased expression, even in the presence of the PGK-puro cassette. The mouse line containing IRES, but not PGK-puro, showed the lowest level of expression. These results suggest that the PGK-puro cassette is necessary to obtain the enhanced expression of a co-existing human cDNA in the mouse Ttr locus, even though the expression of co-existing cDNA was under the control of the mouse endogenous promoter.     

Use of the Cre-loxP recombination system as an estimate for Agrobacterium-mediated co-transformation of tobacco leaves

Agrobacterium tumefaciens-mediated transformation of tobacco leaves (Nicotiana tabacum) is used to study gene expression in a heterologous genetic background. Here, the Cre-loxP recombination system was used to detect T-DNA transfer by two A. tumefaciens cells harboring different binary vectors. Cre, under the control of the CaMV 35S promoter, was cloned into one vector, and a loxP cassette into another vector. A mixture of A. tumefaciens, in which each cell contained either a Cre- or loxP-vector, was co-infiltrated into tobacco leaves. After two days, excision of loxP-flanked DNA was detected by PCR and used as an estimate for co-transformation events. Strongest excision (> 50%) was observed when the loxP cassette was cloned into vector pPZP112 and Cre into pISV2678. This fast and easy technique can be used to assess the co-transformation efficiency of tobacco cells in future studies.     

Excision of the Frt-flanked neo (R) cassette from the CD19cre knock-in transgene reduces Cre-mediated recombination

Intercross of mice transgenic for Flp-recombinase with the CD19cre mouse strain leads to excision of the Frt-flanked neo ^R cassette from the CD19cre knock-in transgene. This significantly reduces the expression level of Cre by the CD19cre transgene and consequently decreases the extent of Cre-mediated recombination of loxP-flanked alleles, most likely due to the fact that this neo ^R cassette contains polyoma enhancer sequences. We wish to draw attention to this finding, since the Flp-deleter mouse strain is commonly used to remove Frt-flanked selection cassettes in vivo from conditional alleles. Therefore conditional alleles have to be separated from the Flp-deleter transgene by breeding before crosses with CD19cre mice are initiated. In addition our findings suggest that gene expression from the CD19 locus can be increased by the insertion of exogenous enhancer sequences, without compromising B cell specificity.     

A short synthetic chimeric sequence harboring matrix attachment region/PSAR2 increases transgene expression in Chinese hamster ovary cells

A chimeric DNA fragment containing an interferon-beta matrix attachment region (MAR) and an immunoglobulin MAR (PSAR2) was synthesized. PSAR2 was cloned into the upstream or downstream region of an enhanced green fluorescent protein (eGFP) expression cassette in a eukaryotic vector, which was then transfected into CHO cells. The results showed that PSAR2 did not effectively increase transgene expression when it was cloned into the upstream region of the eGFP expression cassette. However, when inserted downstream of the eGFP expression cassette, PSAR2-enhanced transient transgene expression and significantly increased the numbers of stably transfected cells compared with the control vector. Additionally, PSAR2 significantly increased eGFP copy numbers as compared with the control vector. PSAR2 could significantly enhance transgene expression in CHO cells according to the position in the vector and increased transgene copy numbers. We found a short chimeric sequence harboring two MARs effectively increased transgene expression in CHO cells.     

Unidirectional Cre-mediated genetic inversion in mice using the mutant loxP pair lox66/lox71

The Cre/loxP recombination system is a commonly used tool to alter the mouse genome in a conditional manner by deletion or inversion of loxP-flanked DNA segments. While Cre-mediated deletion is essentially unidirectional, inversion is reversible and therefore does not allow the stable alteration of gene function in cells that constitutively express Cre. Site-directed mutagenesis yielded a pair of asymmetric loxP sites (lox66 and lox71) that display a favorable forward reaction equilibrium. Here, we demonstrate that lox66/lox71 mediates efficient and predominantly unidirectional inversion of a switch substrate targeted to the mouse genome in combination with either inducible or cell type-specific cre-transgenes in vivo.     

Cre recombinase‐mediated site‐specific modification of a cellular genome using an integrase‐defective retroviral vector

Retroviral integrase is an enzyme responsible for the integration of retroviruses. A single mutation in the integrase core domain can severely compromise its integration ability, leading to the accumulation of circular retroviral cDNA in the nuclei of infected cells. We therefore attempted to use those cDNA as substrates for Cre recombinase to perform a recombinase‐mediated cassette exchange (RMCE), thereby targeting retroviral vectors to a predetermined site. An expression unit containing a promoter, an ATG codon and marker genes (hygromycin resistance gene and red fluorescent protein gene) flanked by wild‐type and mutant loxP sites was first introduced into cellular chromosome to build founder cell lines. We then constructed another plasmid for the production of integrase‐defective retroviral vectors (IDRV), which contains an ATG‐deficient neomycin resistance gene and green fluorescent protein gene, flanked by a compatible pair of loxPs. After providing founder cells with Cre and infecting with IDRV later, effective RMCE occurred, resulting in the appearance of G418‐resistant colonies and a change in the color of fluorescence from red to green. Southern blot and PCR analyses on selected clones further confirmed site‐specific recombination. The successful substitution of the original viral integration machinery with a non‐viral mechanism could expand the application of retroviral vectors. Biotechnol. Bioeng. 2010;107:717–729. © 2010 Wiley Periodicals, Inc.     

Genetic transformation of the codling moth, <Emphasis Type="Italic">Cydia pomonella</Emphasis> L., with <Emphasis Type="Italic">piggyBac</Emphasis> EGFP

Genetic transformation of the codling moth, Cydia pomonella, was accomplished through embryo microinjection with a plasmid-based piggyBac vector containing the enhanced green fluorescent protein (EGFP) gene. Sequencing of the flanking regions around the inserted construct resulted in identification of insect genomic sequences, not plasmid sequences, thus providing evidence that the piggyBac EGFP cassette had integrated into the codling moth genome. EGFP-positive moths were confirmed in the 28th and earlier generations post injection through PCR and Southern blot analyses, indicating heritability of the transgene.