Cre-loxP Site-specific Recombination Research Articles

Targeted insertion of conditional expression cassettes into the mouse genome using the modified i-PITT

BackgroundTransgenic (Tg) mice are widely used in biomedical research, and they are typically generated by injecting transgenic DNA cassettes into pronuclei of one-cell stage zygotes. Such animals often show unreliable expression of the transgenic DNA, one of the major reasons for which is random insertion of the transgenes. We previously developed a method called “pronuclear injection-based targeted transgenesis” (PITT), in which DNA constructs are directed to insert at pre-designated genomic loci. PITT was achieved by pre-installing so called landing pad sequences (such as heterotypic LoxP sites or attP sites) to create seed mice and then injecting Cre recombinase or PhiC31 integrase mRNAs along with a compatible donor plasmid into zygotes derived from the seed mice. PITT and its subsequent version, improved PITT (i-PITT), overcome disadvantages of conventional Tg mice such as lack of consistent and reliable expression of the cassettes among different Tg mouse lines, and the PITT approach is superior in terms of cost and labor. One of the limitations of PITT, particularly using Cre-mRNA, is that the approach cannot be used for insertion of conditional expression cassettes using Cre-LoxP site-specific recombination. This is because the LoxP sites in the donor plasmids intended for achieving conditional expression of the transgene will interfere with the PITT recombination reaction with LoxP sites in the landing pad.ResultsTo enable the i-PITT method to insert a conditional expression cassette, we modified the approach by simultaneously using PhiC31o and FLPo mRNAs. We demonstrate the strategy by creating a model containing a conditional expression cassette at the Rosa26 locus with an efficiency of 13.7%. We also demonstrate that inclusion of FLPo mRNA excludes the insertion of vector backbones in the founder mice.ConclusionsSimultaneous use of PhiC31 and FLP in i-PITT approach allows insertion of donor plasmids containing Cre-loxP-based conditional expression cassettes.

Genome editing of different strains of Aureobasidium melanogenum using an efficient Cre/loxp site-specific recombination system

It has been well known that different strains of Aureobasidium spp. can produce commercial pullulan, polymalate, liamocin, intracellular lipids, gluconic acid, siderophore, melanin and various enzymes. In order to fully elucidate their synthetic pathways and regulation, it is necessary to have an efficient gene editing system for genetic modification of Aureobasidium spp. In this study, an efficient Cre/loxp site-specific recombination system (pAMGDloxp-1, pAMEXlox-1 and pAMCRE1) was constructed. It was found that they could be successfully used to sequentially delete and express many genes in different strains of A. melanogenum. After each round of gene disruption and expression, over 0.5 positive cells per 1000 competent cells and over 49.8 positive transformants per 1.0 μg DNA were achieved. After each round of the antibiotics gene excision by using the Cre-loxp site-specific recombination, over 95.4 % of the antibiotics-resistant cells became sensitive to both hygromycin B and nourseothricin again. This demonstrated that the Cre/loxp site-specific recombination system constructed in this study can efficiently be used to simultaneously delete and express many genes in different strains of A. melanogenum. These systems are promising approaches for the easily modifying genomics of the yeast-like fungal strains with enhanced metabolic pathways through multicopy gene deletion and expression.

TrkB Signaling in Dorsal Raphe Nucleus is Essential for Antidepressant Efficacy and Normal Aggression Behavior.

Brain-derived neurotrophic factor (BDNF) and its high affinity receptor, tropomyosin receptor kinase B (TrkB), have important roles in neural plasticity and are required for antidepressant efficacy. Studies examining the role of BDNF-TrkB signaling in depression and antidepressant efficacy have largely focused on the limbic system, leaving it unclear whether this signaling is important in other brain regions. BDNF and TrkB are both highly expressed in the dorsal raphe nucleus (DRN), a brain region that has been suggested to have a role in depression and antidepressant action, although it is unknown whether BDNF and TrkB in the dorsal raphe nucleus are involved in these processes. We combined the adeno-associated virus (AAV) with the Cre-loxP site-specific recombination system to selectively knock down either Bdnf or TrkB in the DRN. These mice were then characterized in several behavioral paradigms including measures of depression-related behavior and antidepressant efficacy. We show that knockdown of TrkB, but not Bdnf, in the DRN results in loss of antidepressant efficacy and increased aggression-related behavior. We also show that knockdown of TrkB or Bdnf in this brain region does not have an impact on weight, activity levels, anxiety, or depression-related behaviors. These data reveal a critical role for TrkB signaling in the DRN in mediating antidepressant responses and normal aggression behavior. The results also suggest a non-cell autonomous role for BDNF in the DRN in mediating antidepressant efficacy.

Reversibility of the Snail-induced epithelial–mesenchymal transition revealed by the Cre–loxP system

The epithelial–mesenchymal transition (EMT), a key process in the tumor metastatic cascade, is characterized by the loss of cell–cell junctions and cell polarity, as well as the acquisition of migratory and invasive properties. Snail is an EMT-inducer whose expression in several different epithelial cells, e.g., Madin–Darby canine kidney (MDCK), leads to EMT. To further understand EMT induced by Snail expression, the Cre–loxP site-specific recombination system was used to investigate its reversibility. Transfection of MDCK cells with loxP-flanked Snail (Snail-loxP) resulted in EMT induction, which included the acquisition of a spindle-shaped fibroblastic morphology, the downregulation of epithelial markers, and the upregulation of mesenchymal markers. DNA methylation of the E-cadherin promoter, which often occurs during E-cadherin downregulation, was not observed in Snail+ cells. After Cre-mediated excision of Snail-loxP, the cells reacquired an epithelial morphology, upregulated epithelial markers, and downregulated mesenchymal markers. Thus, EMT induced by Snail expression was reversible.

Development of pronuclear injection-based targeted transgenesis in mice through Cre-loxP site-specific recombination.

Microinjection of DNA into the pronuclei of zygotes is the simplest and most widely used method for generating transgenic (Tg) mice. However, it is always associated with random integration of multiple copies of the transgene, resulting in unstable, low, or no transgene expression due to positional effects and/or repeat-induced gene silencing. In addition, random integration sometimes disrupts an endogenous gene that can affect the phenotypes of Tg mice. Our recently developed pronuclear injection-based targeted transgenesis (PITT) method enables the integration of a single-copy transgene into a predetermined genomic locus through Cre-loxP site-specific recombination. The PITT method enables stable and reliable transgene expression in Tg mice and is also applicable for generating knockdown mice. Therefore, the PITT method could represent next-generation transgenesis that overcomes the pitfalls of conventional transgenesis.

Gene Editing a Constitutively Active OsRac1 by Homologous Recombination-Based Gene Targeting Induces Immune Responses in Rice

OsRac1 is a member of the plant small GTPase Rac/Rop family and plays a key role in rice immunity. The constitutively active (CA) G19V mutation of OsRac1 was previously shown to induce reactive oxygen species production, phytoalexin synthesis and defense gene activation, leading to resistance to rice blast infection. To study further the effect of the G19V mutation in disease resistance, we introduced a single base substitution by gene targeting and removed the selectable marker using Cre-loxP site-specific recombination. The CA-OsRac1 gene generated by gene targeting was termed CA-gOsRac1. The G19V mutation was transferred from a targeting vector to the OsRac1 locus and stably transmitted to the next generation. In the leaf blade of homozygous CA-gOsRac1 plants, mutant transcript levels were much lower than in those of wild-type plants. In contrast, mutant transcripts in roots, leaf sheaths and panicles were more abundant than those in leaf blades. However, upon chitin treatment, the expression of defense-related genes PAL1 and PBZ1 in the cell culture was greater in the mutants compared with wild-type plants. Furthermore, induction of hypersensitive response (HR)-like cell death was observed in the leaf sheaths of mutant plants infected with a compatible race of rice blast fungus. In the CA-gOsRac1 plants, a number of genes previously shown to be induced by Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae (Xoo) infection were induced in the leaf sheath without pathogen infection. These results suggest that gene targeting will provide mutations useful for gene function studies and crop improvement.

Synapsis of loxP Sites by Cre Recombinase

Cre recombinase catalyzes site-specific recombination between 34-bp loxP sites in a variety of topological and cellular contexts. An obligatory step in the recombination reaction is the association, or synapsis, of Cre-bound loxP sites to form a tetrameric protein assembly that is competent for strand exchange. Using analytical ultracentrifugation and electrophoresis approaches, we have studied the energetics of Cre-mediated synapsis of loxP sites. We found that synapsis occurs with a high affinity (Kd = 10 nM) and is pH-dependent but does not require divalent cations. Surprisingly, the catalytically inactive Cre K201A mutant is fully competent for synapsis of loxP sites, yet the inactive Y324F and R173K mutants are defective for synapsis. These findings have allowed us to determine the first crystal structures of a pre-cleavage Cre-loxP synaptic complex in a configuration representing the starting point in the recombination pathway. When combined with a quantitative analysis of synapsis using loxP mutants, the structures explain how the central 8 bp of the loxP site are able to dictate the order of strand exchange in the Cre system.

Vascular remodelling in the absence and presence of cGKI

The signaling molecule nitric oxide (NO) is of crucial importance for smooth muscle cell (SMC) function and exerts numerous, sometimes opposing effects on vascular proliferative diseases, such as atherosclerosis and restenosis. Under physiological conditions many effects of NO are mediated via the cyclic guanosine-3',5'-monophosphate (cGMP)/cGMP-dependent protein kinase type I (cGKI) pathway. In the cardiovascular system protective effects of NO signaling have been generally attributed to cGMP/cGKI. In contrast, novel genetic evidence from the analysis of atherosclerosis-prone mice indicated that endogenous cGKI accelerated plaque growth and, thus, was pro-atherogenic. However, in restenosis after intraarterial interventions, such as balloon angioplasty or stent placement the mediator(s) of NO signals, in particular the role of endogenous SMC cGKI is unclear. Towards a better understanding of cGKI and its function in restenosis the Cre-loxP site-specific recombination system was used to generate conditional cardiac and SMC cGKI knockout mice. To test whether the lack of cGKI in SMCs affects the injury-induced vascular remodelling conditional mutants and control mice were analysed by morphometry and immunohistochemistry at different time points after injury, in a normolipidemic situation as well as on an apolipoprotein E-deficient genetic background. In addition, by continuous administration of sildenafil the cGMP/cGKI pathway was chronically activated in wild type mice subjected to the injury.

Toward simpler and faster genome-wide mutagenesis in mice

Here we describe a practical Cre-loxP and piggyBac transposon-based mutagenesis strategy to systematically mutate coding sequences and/or the vast noncoding regions of the mouse genome for large-scale functional genomic analysis. To illustrate this approach, we first created loxP-containing loss-of-function alleles in the protocadherin alpha, beta and gamma gene clusters (Pcdha, Pcdhb and Pcdhg). Using these alleles, we show that, under proper guidance, Cre-loxP site-specific recombination can mediate efficient trans-allelic recombination in vivo, facilitating the generation of large germline deletions and duplications including deletions of Pcdha, and Pcdha to Pcdhb, simply by breeding (that is, at frequencies of 5.5%-21.6%). The same breeding method can also generate designed germline translocations between nonhomologous chromosomes at unexpected frequencies of greater than 1%. By incorporating a piggyBac transposon to insert and to distribute loxP sites randomly throughout the mouse genome, we present a simple but comprehensive method for generating genome-wide deletions and duplications, in addition to insertional loss-of-function and conditional rescue alleles, again simply by breeding.

A passage through in vitro culture leads to efficient production of marker-free transgenic plants in Brassica juncea using the Cre–loxP system

The Cre-loxP site-specific recombination system was deployed for removal of marker genes from Brassica juncea (Indian mustard). Excision frequencies, monitored by removal of nptII or gfp genes in F(1) plants of crosses between LOX and CRE lines, were high in quiescent, differentiated somatic tissues but extremely poor in the meristematic regions (and consequently the germinal cells) thus preventing identification and selection of marker-free transgenic events which are devoid of both the marker gene as well as the cre gene, in F(2) progeny. We show that a passage through in vitro culture of F(1 )leaf explants allows efficient development of marker-free transgenics in the F(2) generation addressing current limitations associated with efficient use of the Cre/loxP technology for marker gene removal.

Construction of marker-free transplastomic tobacco using the Cre-loxP site-specific recombination system

Incorporation of a selectable marker gene in the plastid genome is essential to uniformly alter the thousands of genome copies in a tobacco cell. When transformation is accomplished, however, the marker gene becomes undesirable. Here we describe plastid transformation vectors, the method of plastid transformation using tobacco leaves and alternative protocols for marker gene excision with the P1 bacteriophage Cre-loxP site-specific recombination system. Plastid vectors carry a marker gene flanked with directly oriented loxP sites and a gene of interest, which are introduced into plastids by the biolistic process. The transforming DNA integrates into the plastid genome by homologous recombination via plastid targeting sequences. Marker gene excision is accomplished by a plastid-targeted Cre protein expressed from a nuclear gene. Expression may be from an integrated gene introduced by Agrobacterium transformation (Transformation Protocol), by pollination (Pollination Protocol) or from a transient, non-integrated T-DNA (Transient Protocol). Transplastomic plants are obtained in about 3 months, yielding seed after 2 months. The time required to remove the plastid marker and nuclear genes and to obtain seed takes 10-16 months, depending on which protocol is used.

Preferential synapsis of loxP sites drives ordered strand exchange in Cre-loxP site-specific recombination

The bacteriophage P1 Cre recombinase catalyzes site-specific recombination between 34-base-pair loxP sequences in a variety of topological contexts. This reaction is widely used to manipulate DNA molecules in applications ranging from benchtop cloning to genome modifications in transgenic animals. Despite the simple, highly symmetric nature of the Cre-loxP system, there is strong evidence that the reaction is asymmetric; the 'bottom' strands in the recombining loxP sites are preferentially exchanged before the 'top' strands. Here, we address the mechanistic basis for ordered strand exchange in the Cre-loxP recombination pathway. Using suicide substrates containing 5'-bridging phosphorothioate linkages at both cleavage sites, fluorescence resonance energy transfer between synapsed loxP sites and a Cre mutant that can cleave the bridging phosphorothioate linkage but not a normal phosphodiester linkage, we showed that preferential formation of a specific synaptic complex between loxP sites imposes ordered strand exchange during recombination and that synapsis stimulates cleavage of loxP sites.

The Cre-loxP recombination-based reporter system for plant transcriptional expression studies*

To facilitate the characterization of plant genes, the Cre-loxP site-specific recombination system was adapted to make reporter vectors for plant expression studies. This system allows promoter fragments to be cloned into a small vector (univector) and subsequently recombined in vitro with binary vectors containing different reporter genes precisely at near-perfect efficiency. We have constructed univector-adapted vectors with three reporters, beta-glucuronidase, luciferase, and green fluorescent protein, and a BASTA-resistance gene for selection of plant transformants. Expression in plants using the new system was validated by comparison to conventional reporter vectors. These new vectors are efficient and economical alternatives to the other plant reporter vectors currently available. The royalty-free Cre-loxP system serves as a platform for the future expansion of recombination-based cloning vectors for plant research.

The testicular fatty acid binding protein PERF15 regulates the fate of germ cells in PERF15 transgenic mice

The quality control of sperm is critical for efficient reproduction. In germ cells, cell death involves different processes to those in somatic cells, and in many cases, the trigger to induce cell death in deficient germ cells is still unclear. It is known that the fatty acid composition of sperm is related to fertility. Composition of the fatty acid of germ cells changes dynamically during spermatogenesis, and fatty acid binding protein (FABP) may be involved in these changes. In this study, we developed transgenic mice with a testicular germ-cell-specific FABP (PERF15) transgene, whose expression was controlled by the Cre-LoxP site-specific recombination system. We also developed transgenic mice with the Cre gene under the control of the spermatocyte specific Pgk2 promoter. In double transgenic mice, following Cre-mediated recombination of the PERF15 containing transgene, PERF15 was strongly overexpressed. Its overexpression induced multinucleate symplasts to form, indicating programmed germ cell death occurred at the elongated spermatid stage. As a result, sperm harboring the transgene were significantly decreased, but the surviving sperm demonstrated higher fertility than natural sperm. Therefore, we conclude that PERF15 associate with the direction of germ cell fates and preserve the quality of sperm.

Baculovirus expression system for heterologous multiprotein complexes

The discovery of large multiprotein complexes in cells has increased the demand for improved heterologous protein production techniques to study their molecular structure and function. Here we describe MultiBac, a simple and versatile system for generating recombinant baculovirus DNA to express protein complexes comprising many subunits. Our method uses transfer vectors containing a multiplication module that can be nested to facilitate assembly of polycistronic expression cassettes, thereby minimizing requirements for unique restriction sites. The transfer vectors access a modified baculovirus DNA through Cre-loxP site-specific recombination or Tn7 transposition. This baculovirus has improved protein expression characteristics because specific viral genes have been eliminated. Gene insertion reactions are carried out in Escherichia coli either sequentially or concurrently in a rapid, one-step procedure. Our system is useful for both recombinant multiprotein production and multigene transfer applications.

A reliable lacZ expression reporter cassette for multipurpose, knockout-first alleles.

Alteration of the mouse genome through homologous recombination in embryonic stem (ES) cells is the most accurate and versatile way to dissect gene function in a vertebrate model. Most often, a selectable marker is used to create a knockout allele by replacing an essential part of the gene. However, knockout strategies are limited because the mutation is present constitutively. Conditional approaches based on the Cre-loxP site-specific recombination (SSR) system address this limitation; however, it requires that all parts of the targeted gene remain in ES cells. Here we report success with a "knockout-first" strategy that ablates gene function by insertion of RNA processing signals without deletion of any of the target gene. Incorporation of site-specific recombination target sites creates a multipurpose allele for both knockout and conditional applications.

Conditional, recombinase-mediated expression of genes in plant cell cultures.

In plant cells, overexpression of critical genes can be hampered by deleterious effects on development that results in a counterselection of transgenic cells harboring the gene of interest. Inducible expression systems have been reported, but many of them show unwanted leaky expression. To circumvent this potential problem, a novel inducible system was developed based on two previously characterized systems: the CRE-loxP site-specific recombination system of bacteriophage P1 and the subcellular targeting of proteins by a mammalian glucocorticoid receptor (GR). By fusing the receptor domain of the rat GR to the carboxyl terminus of the CRE recombinase, a double-lock conditional transcriptional induction system was created that is highly useful to overexpress genes whose expression may block transgenic regeneration. Furthermore, because the designed vector utilizes the GATEWAY recombination technology, cloning was restriction- and ligation-free, thus rendering the vector suitable for high-throughput research. The system was tested in Nicotiana tabacum bright yellow-2 (BY-2) cells and its efficiency was demonstrated for the controlled overexpression of the gus reporter gene and a mutant allele of the A-type cyclin-dependent kinase (CDKA), which is known to be a potent inhibitor of the cell cycle.

High efficiency, site-specific excision of a marker gene by the phage P1 cre-loxP system in the yellow fever mosquito, Aedes aegypti.

The excision of specific DNA sequences from integrated transgenes in insects permits the dissection in situ of structural elements that may be important in controlling gene expression. Furthermore, manipulation of potential control elements in the context of a single integration site mitigates against insertion site influences of the surrounding genome. The cre-loxP site-specific recombination system has been used successfully to remove a marker gene from transgenic yellow fever mosquitoes, Aedes aegypti. A total of 33.3% of all fertile families resulting from excision protocols showed evidence of cre-loxP-mediated site-specific excision. Excision frequencies were as high as 99.4% within individual families. The cre recombinase was shown to precisely recognize loxP sites in the mosquito genome and catalyze excision. Similar experiments with the FLP/FRT site-specific recombination system failed to demonstrate excision of the marker gene from the mosquito chromosomes.

Improved packaging system for generation of high-level noncytotoxic HSV-1 amplicon vectors using Cre-loxP site-specific recombination to delete the packaging signals of defective helper genomes.

Amplicons are promising helper-dependent HSV-1-derived vectors that allow the transfer and expression of very large foreigner DNA into dividing and quiescent cells. We had already described an approach to prepare large amounts of high-titer amplicon vectors, using Cre-loxP site-specific recombination system to delete the packaging ("a") signals of an HSV-1 recombinant helper virus (HSV-1 LaL). Amplicon vectors prepared using such a system showed a level of contamination with helper particles lower than 1%. The residual helper particles generated by this system are, however, replication-competent, thus precluding their use in gene therapy. To avoid such potential spread of residual particles, we present here the development of a defective Cre-loxP-based helper virus (HSV-1 LaL Delta J), deleted of the genes encoding ICP4 and ICP34.5 proteins from the helper genome, in addition to the native "a" signals. HSV-1 LaL Delta J carries a single floxed "a" signal in gC locus. To produce HSV-1 LaL Delta J and to prepare the amplicon vectors, we have constructed two novel cell lines expressing the essential ICP4 protein, either alone or in combination with Cre recombinase. These cell lines were conceived to complement ICP4 while minimizing the probability of generating replication-competent particles. In this paper we present results demonstrating that the novel helper system allows ready production of large amounts of high-titer amplicon vectors. Residual helper particles generated still do not exceed 0.5% of the viral population and can grow only in cells expressing ICP4. Amplicon vectors produced with this method showed no cytotoxicty for infected cells.

Conditional expression of UAS-transgenes in the adult eye with a new gene-switch vector system.

The spatial control of transgene expression with the Gal4 binary system has been one of the most productive experimental techniques applied to Drosophila melanogaster (Brand and Perrimon, 1993). However, the inability to temporally regulate the timing of gene expression can frequently make some experiments involving later developmental stages extremely difficult or even unachievable. Also, the lack of experimenter’s control over the induction of gene expression usually makes withingenotype controls impossible. To overcome these problems, the Gal4 system has been modified to permit both spatial and temporal control of transgene expression by the use of fusions proteins containing the Gal4 DNA binding domain and steroid receptor ligand binding domains (Han et al., 2000; Osterwalder et al., 2001; Roman et al., 2001). We have made a new Drosophila vector system that is based on the Gene-Switch fusion protein (Wang et al., 1994). These vectors are designed to facilitate the rapid cloning of promoters and enhancers in front of the Gene-Switch coding region. We have used these vectors to generate lines in which Gene-Switch expression is controlled by the Glass Multiple Repeat enhancer (GMR) from the ninaE regulatory region (Ellis et al., 1993). These P{GMRGS} lines provide inducible, eye-delimited expression of UAS transgenes. The Gene-Switch fusion protein is composed of the Sacchromyces cerivisiae Gal4 DNA binding domain, the human progesterone receptor ligand binding domain, and the human p65 transcriptional activation domain (Burcin et al., 1998; Wang et al., 1994). Antiprogestins, such as RU486, will bind to the progesterone receptor ligand binding domain and activate the Gene-Switch molecule. Once activated, Gene-Switch will promote transcription from the UAS Gal4-regulated promoters. The Gene-Switch protein, when expressed in Drosophila, has almost undetectable activity in the absence of RU486; however, feeding RU486 to Gene-Switch expressing flies leads to a dramatic induction of transcription from Gal4-regulated promoters (Osterwalder et al., 2001; Roman et al., 2001). Gene-Switch is responsive to RU486 in a dose-dependent manner and feeding flies 500 M RU486 for 1 h appears to saturate the response (Osterwalder et al., 2001; Roman et al., 2001). GeneSwitch-dependent activation can be detected in as little as 3 h and reaches maximum levels at approximately 24 h after the initial feeding. Thus, the timing and level of transcription of a UAS transgene can be manipulated through the controlled feeding of RU486. We have previously used enhancer detector constructs to achieve spatially restricted expression of the Gene-Switch fusion protein in a number of tissue and cell types (Roman et al., 2001). Additionally, both the Myosin Heavy Chain and elav promoters have been fused to the Gene-Switch coding sequences to provide, respectively, muscle and nervous system-specific expression in Drosophila (Osterwalder et al., 2001). Nevertheless, there remain many interesting and important cells and tissues for which we do not yet have Gene-Switch expressing lines. Further defined promoters and enhancers should be an important source for these desirable lines. Therefore, we have made a series of new P-elements that can be used to identify new transcriptional regulatory sequences through their ability to drive GeneSwitch expression. The new Gene-Switch P-elements allow for the rapid and efficient cloning of enhancers and promoters in front of the Gene-Switch coding sequences. A common problem with cloning into P-element vectors is the lack of unique restriction sites. Additionally, the need to clone many different DNA fragments into several different vectors can take a significant amount of time and effort in both the planning and cloning. The new vectors are an adaptation of the Univector plasmid-fusion system, which utilizes the cre-loxP site-specific recombination system of phage P1 (Liu et al., 1998). The Univector P-element system is composed of a donor plasmid and an acceptor P-element (Fig. 1). After the enhancer or promoter sequence is cloned into the multiple cloning sequence of the donor plasmid, cre-mediated in vitro recombination will fuse the two plasmids such that the