FLP recombinase-mediated site-specific recombination in silkworm, Bombyx mori

A comprehensive understanding of gene function and the production of site-specific genetically modified mutants are two major goals of genetic engineering in the post-genomic era. Although site-specific recombination systems have been powerful tools for genome manipulation of many organisms, they have not yet been established for use in the manipulation of the silkworm Bombyx mori genome. In this study, we achieved site-specific excision of a target gene at predefined chromosomal sites in the silkworm using a FLP/FRT site-specific recombination system. We first constructed two stable transgenic target silkworm strains that both contain a single copy of the transgene construct comprising a target gene expression cassette flanked by FRT sites. Using pre-blastoderm microinjection of a FLP recombinase helper expression vector, 32 G3 site-specific recombinant transgenic individuals were isolated from five of 143 broods. The average frequency of FLP recombinase-mediated site-specific excision in the two target strains genome was approximately 3.5%. This study shows that it is feasible to achieve site-specific recombination in silkworms using the FLP/FRT system. We conclude that the FLP/FRT system is a useful tool for genome manipulation in the silkworm. Furthermore, this is the first reported use of the FLP/FRT system for the genetic manipulation of a lepidopteran genome and thus provides a useful reference for the establishment of genome manipulation technologies in other lepidopteran species.     

Developing a flippase-mediated maker recycling protocol for the oleaginous yeast <Emphasis Type="Italic">Rhodosporidium toruloides</Emphasis>

Objectives

To establish a recombinase flippase (FLP) and flippase recognition target (FRT) system-mediated protocol for post-integration excision of exogenous DNA fragments in the oleaginous yeast Rhodosporidium toruloides.

Results

Binary vectors were constructed to harbor FLP expressing cassette together with the hygromycin-resistance marker. Results showed that R. toruloides transformants produced FLP, but failed to mediate removal of the bleomycin-resistance marker within two FRT sites. When FLP was fused with a native nuclear localization signal (NLS) peptide, the system was found functional. Moreover, R. toruloides recombinant strains expressing the NLS-fused FLP under the control of PADH2, an promoter of alcohol dehydrogenase 2 gene (RHTO_03062), were obtained to realize homologous recombination upon growing in glucose-deficient medium.

Conclusions

We have devised a homologous recombination method for R. toruloides based on the FLP/FRT system, which may facilitate further metabolic engineering and designing advanced cell factories for value-added chemicals.

     

FLP-mediated site-specific recombination in microinjected murine zygotes

The FLP recombinase of yeast catalyses site-specific recombination between repeated FLP recombinase target (FRT) elements in yeast and in heterologous system (Escherichia coli, Drosophila, mosquito and cultured mammalian cells). In this report, it is shown that transient FLP recombinase expression can recombine and activate an extrachromosomal silent reporter gene following coinjection into fertilized one-cell mouse eggs. Furthermore, it is demonstrated that introduction of a FLP-recombinase expression vector into transgenic one-cell fertilized mouse eggs induces a recombination event at a chromosomal FRT target locus. The resulting event occured at the one-cell stage and deleted a chromosomal tandem array of a FRT containinglacZ expression cassette down to one or two copies. These results demonstrate that the FLP recombinase can be utilized to manipulate the genome of transgenic animals and suggest that FLP recombinase-mediated plasmid-to-chromosome targeting is feasible in microinjected eggs.     

Elimination of marker genes and targeted integration via FLP/<Emphasis Type="BoldItalic">FRT</Emphasis> recombination system from yeast in hybrid aspen (<Emphasis Type="BoldItalic">Populus tremula</Emphasis> L. × <Emphasis Type="Italic">P. tremuloides</Emphasis> Michx.)

Marker gene elimination was investigated in hybrid aspen (Populus tremula L. × Populus tremuloides Michx.) using the FLP/FRT recombination system. The construct contained the FLP recombinase under control of a heat inducible promoter, the antibiotic resistance gene nptII driven by the CaMV 35S promoter, and a promoterless uidA gene. The construct was integrated into poplar via Agrobacterium-mediated transformation. The active FLP recombinase excised the nptII marker gene and combined the promoterless uidA gene with the CaMV 35S promoter to form an active uidA gene. For targeted transgene integration, two constructs were used. The first one carried FLP under control of the heat-inducible Gmhsp17.5-E promoter from soybean as well as an active nptII gene flanked by two FRT sites; the second contained the promoterless bar selection marker gene also flanked by two FRT sites. Following transformation and induction of FLP, the enzyme mediated a site-specific recombination at the FRT sites of both constructs. This recombination leads to an excision of the FLP and nptII gene from the first as well as an excision of the promoterless bar gene from the second construct. The promoterless bar gene reintegrated exactly at the former position of the FLP and nptII genes in the first construct to form an active bar gene. The FLP/FRT recombination system from yeast forms a promising basis for the production of antibiotic-free transgenic plants and a useful tool for directed integration of transgenes into plant genomes.     

Site-Specific Integration of Transgenes in Soybean via Recombinase-Mediated DNA Cassette Exchange

A targeting method to insert genes at a previously characterized genetic locus to make plant transformation and transgene expression predictable is highly desirable for plant biotechnology. We report the successful targeting of transgenes to predefined soybean (Glycine max) genome sites using the yeast FLP-FRT recombination system. First, a target DNA containing a pair of incompatible FRT sites flanking a selection gene was introduced in soybean by standard biolistic transformation. Transgenic events containing a single copy of the target were retransformed with a donor DNA, which contained the same pair of FRT sites flanking a different selection gene, and a FLP expression DNA. Precise DNA cassette exchange was achieved between the target and donor DNA via recombinase-mediated cassette exchange, so that the donor DNA was introduced at the locus previously occupied by the target DNA. The introduced donor genes expressed normally and segregated according to Mendelian laws.Plant transformation has challenges such as random integration, multiple transgene copies, and unpredictable expression. Homologous recombination (Iida and Terada, 2005; Wright et al., 2005) and DNA recombinase-mediated site-specific integration (SSI) are promising technologies to address the challenges for placing a single copy of transgenes into a precharacterized site in a plant genome.Several site-specific DNA recombination systems, such as the bacteriophage Cre-lox and the yeast FLP-FRT and R-RS, have been used in SSI studies (Ow, 2002; Groth and Calos, 2003). A common feature of these systems is that each system consists of a recombinase Cre, FLP, or R and two identical or similar palindromic recognition sites, lox, FRT, or RS. Each recognition site contains a short asymmetric spacer sequence where DNA strand exchange takes place, flanked by inverted repeat sequences where the corresponding recombinase specifically binds. If two recognition sites are located in cis on a DNA molecule, the DNA segment can be excised if flanked by two directionally oriented sites or inverted if flanked by two oppositely oriented sites. If two recognition sites are located in trans on two different DNA molecules, a reciprocal translocation can happen between the two DNA molecules or the two molecules can integrate if at least one of them is a circular DNA (Ow, 2002; Groth and Calos, 2003).Single-site SSI can integrate a circular donor DNA containing one recognition site into a similar site previously placed in a plant genome. The integrated transgene now flanked by two recognition sites is vulnerable to excision. Transient Cre expression and the use of mutant lox sites to create two less compatible sites after integration helped reduce the subsequent excision in tobacco (Nicotiana tabacum; Albert et al., 1995; Day et al., 2000). A similar approach was used to produce SSI events in rice (Oryza sativa), and the transgene was proven stably expressed over generations (Srivastava and Ow, 2001; Srivastava et al., 2004; Chawla et al., 2006). Using a promoter trap to displace a cre gene in the genome with a selection gene from the donor, approximately 2% SSI was achieved in Arabidopsis (Arabidopsis thaliana; Vergunst et al., 1998).When two recognition sites located on a linear DNA molecule are similar enough to be recognized by the same recombinase but different enough to reduce or prevent DNA recombination from happening between them, the DNA segment between the two sites may not be easily excised or inverted. When a circular DNA molecule carrying the same two incompatible sites is introduced, the circular DNA can integrate by the corresponding recombinase at either site on the linear DNA to create a collinear DNA with four recognition sites, two from the original linear DNA and two from the circular DNA. DNA excision can subsequently occur between any pair of compatible sites to restore the two original DNA molecules or to exchange the intervening DNA segments between the two DNA molecules. This process, termed recombinase-mediated cassette exchange (RMCE), can be employed to integrate transgenes directionally into predefined genome sites (Trinh and Morrison, 2000; Baer and Bode, 2001).RMCE using two oppositely oriented identical RS sites, a donor containing the R recombinase gene and a third RS site to limit random integration, resulted in cassette exchange between the donor and a previously placed target in tobacco (Nanto et al., 2005). RMCE using both the Cre-lox and FLP-FRT systems improved RMCE frequency in animal cell cultures (Lauth et al., 2002). RMCE using two directly oriented incompatible FRT sites and transiently expressed FLP recombinase achieved cassette exchange between a target previously placed in the Drosophila genome and a donor introduced as a circular DNA (Horn and Handler, 2005). A gene conversion approach involving Cre-lox- and FLP-FRT-mediated SSI, RMCE, and homologous recombination was explored in maize (Zea mays; Djukanovic et al., 2006). RMCE using two oppositely oriented incompatible lox sites and transiently expressed Cre recombinase produced single-copy RMCE plants in Arabidopsis (Louwerse et al., 2007).To develop FLP-FRT-mediated RMCE in soybean (Glycine max), we created transgenic target lines containing a hygromycin resistance gene flanked by two directly oriented incompatible FRT sites via biolistic transformation. Single-copy target lines were selected and retransformed with a donor DNA containing a chlorsulfuron resistance gene flanked by the same pair of FRT sites. An FLP expression DNA was cobombarded to transiently provide FLP recombinase. RMCE events were obtained from multiple target lines and confirmed by extensive molecular characterization.     

Effect of <Emphasis Type="Italic">BBX-B8</Emphasis> overexpression on development,body weight,silk protein synthesis and egg diapause of <Emphasis Type="Italic">Bombyx mori</Emphasis>

Bombyxin (BBX) is an insulin-like peptide exists in the silkworm Bombyx mori. Our previous studies on the effects of inhibiting BBX-B8 expression found that BBX-B8 is important for the development of organ, reproduction and trehalose metabolism in the silkworms. In this paper, we investigated the expression profile of the BBX-B8 gene and effect of BBX-B8 overexpression on the development, body weight, silk protein synthesis and egg diapause of B. mori to further understand BBX-B8 functions. BBX-B8 gene expression could be detected in the brains, midguts, anterior silkglands, ovaries, testes, fat bodies, hemolymph, malpighian tubules and embryos by RT-PCR, however it was mainly expressed in the brain. Western blots showed that the change in BBX-B8 expression was not obvious in the brain of 1- to 4-day-old larvae of fifth instar silkworms, but expression increased substantially at 5- to 6-day-old larvae of fifth instar silkworms. Transgenic silkworms overexpressing BBX-B8 were obtained by introducing non-transposon transgenic vector pIZT-B8 containing a BBX-B8 gene driven by Orgyia pseudotsugata nucleopolyhedrovirus IE2 promoter into the genome. Development duration of the transgenic silkworms was delayed by 2.5–3.5 days. Cocoon shell weight of transgenic silkworms was reduced by 4.79 % in females and 7.44 % in males, pupal weight of transgenic silkworms was reduced 6.75 % in females and 13.83 % in males compared to non-transgenic silkworms, and 5.56–14.29 % of transgenic moths laid nondiapausing eggs. All results indicated that BBX-B8 plays an important role in the development, silk protein synthesis and egg diapause of silkworm.     

Improved FLP Recombinase,FLPe, Efficiently Removes Marker Gene from Transgene Locus Developed by Cre–<Emphasis Type="Italic">lox</Emphasis> Mediated Site-Specific Gene Integration in Rice

Site-specific recombination systems, such as FLP–FRT and Cre–lox, carry out precise recombination reactions on their respective targets in plant cells. This has led to the development of two important applications in plant biotechnology: marker-gene deletion and site-specific gene integration. To draw benefits of both applications, it is necessary to implement them in a single transformation process. In order to develop this new process, the present study evaluated the efficiency of FLP–FRT system for excising marker gene from the transgene locus developed by Cre–lox mediated site-specific integration in rice. Two different FLP recombinases, the wild-type FLP (FLPwt) and its thermostable derivative, FLPe, were used for the excision of marker gene flanked by FLP recombination targets (FRT). While marker excision mediated by FLPwt was undetectable, use of FLPe resulted in efficient marker excision in a number of transgenic lines, with the relative efficiency reaching up to ~100%. Thus, thermo-stability of FLP recombinase in rice cells is critical for efficient site-specific recombination, and use of FLPe offers practical solutions to FLP–FRT-based biotechnology applications in plants.     

Development of a heat shock inducible and inheritable RNAi system in silkworm

A heat shock inducible and inheritable RNA interference (RNAi) system was developed in the silkworm (Bombyx mori). RNAi transgenic silkworms were generated by injecting silkworm eggs with a piggyBac transposon plasmid carrying RNAi sequence against target gene driven by the Drosophila heat shock protein 70 (HSP70) promoter and the helper plasmid expressing piggyBac transposase. The transgenic EGFP gene and the endogenous eclosion hormone (EH) gene were chosen respectively as the target genes. In the RNAi transgenic silkworms, heat shock at 42 degrees C significantly and specifically reduced the expression of EGFP or EH gene in silkworms according to the corresponding RNAi targeting sequence but not in silkworms with the irrelevant RNAi sequence demonstrating the efficiency and specificity of the RNAi effect. Heat shock in the pupal stage hampered pupal-adult eclosion and reduced egg fertility in EH RNAi transgenic silkworms but not in the wild type or EGFP RNAi transgenic silkworms. The establishment of this heat inducible and inheritable conditional RNA interference system in silkworms provided an approach for the first time to dissect the functions of target genes in silkworms at different stages.     

FLP/FRT-mediated restoration of normal phenotypes and clonal sectors formation in rolC transgenic tobacco

Site-specific recombination systems have been shown to excise transgene DNA sequences positioned between their cognate target sites, and thus be used to generate clonal sectors in transgenic plants. Here we characterized clonal sectors derived from genetic reversion of rolC (A. rhizogenes) – induced vegetative and reproductive phenotypes, mediated by FLP recombinase from S. cerevisiae, in tobacco. The constitutive expression of rolC induces pleiotropic effects including reduced apical dominance and plant height, lanceolate and pale green leaves and small, male-sterile flowers. Two transgenic male-sterile tobacco lines (N. tabacum, Samsun NN) expressing a 35sP-rolC gene construct flanked by two FRT (FLP recombinase target) sites, were cross-pollinated with pollen from a constitutive 35sP-FLP expressing line. Three main phenotypes were generated in result of recombinase-mediated excision of the 35sP-rolC locus in the F₁ (FLP×FRT-35sP-rolC-FRT) hybrid progenies: (a) restoration of male fertility, associated with reversion to normal leaf phenotypes prior to flower bud formation, (b) development of normal and fertile lateral shoot sectors on the background of rolC-type plants, (c) restoration of partially fertile flowers, associated with display of peripheral normal leaf sectors surrounding rolC-type inner-leaf tissues, consistent with periclinal chimeras. These results, supported by DNA molecular analysis, indicate that site-specific recombination might be used as a relatively efficient tool for generation of transgenic periclinal chimeric plants.     

A mini-Mu transposon-based method for multiple DNA fragment integration into bacterial genomes

A method for construction of bacterial strains with multiple DNA inserted into their chromosomes has been developed based on the mini-Mu transposon and FLP/FRT recombination. Exogenous DNA can be integrated by Mu transposition with an FRT cassette containing selection marker and conditional replicative origin (R6Kγori). Subsequently, with the introduction of a helper plasmid bearing gene of FLP recombinase, drug-resistant selection marker is excised from the chromosome. Cells cured of the helper plasmid can undergo the next cycle of transposition and excision of selection marker. Each cycle can add further foreign gene(s) to the chromosome. As an example, resistance genes of chloramphenicol, tetracycline, and gentamicin were successively integrated into the chromosome of Escherichia coli BW25113 by three cycles of insertion and excision as described above. This method proved to be simple and time-saving, which could be applicable to a variety of microorganisms.     

In vivo site-specific integration of transgene in silkworm via PhiC31 integrase-mediated cassette exchange

Current techniques for genetic engineering of the silkworm Bombyx mori genome utilize transposable elements, which result in positional effects and insertional mutagenesis through random insertion of exogenous DNA. New methods for introducing transgenes at specific positions are therefore needed to overcome the limitations of transposon-based strategies. Although site-specific recombination systems have proven powerful tools for genome manipulation in many organisms, their use has not yet been well established for the integration of transgenes in the silkworm. We describe a method for integrating target genes at pre-defined chromosomal sites in the silkworm via phiC31/att site-specific recombination system-mediated cassette exchange. Successful recombinase-mediated cassette exchange (RMCE) was observed in the two transgenic target strains with an estimated transformation efficiency of 3.84–7.01%. Our results suggest that RMCE events between chromosomal attP/attP target sites and incoming attB/attB sites were more frequent than those in the reciprocal direction. This is the first report of in vivo RMCE via phiC31 integrase in the silkworm, and thus represents a key step toward establishing genome manipulation technologies in silkworms and other lepidopteran species.     

Germ line transformation of the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>, using the transposable element <Emphasis Type="Italic">Minos</Emphasis>

We investigated the use of Minos as a vector for transgenesis in the silkworm, Bombyx mori. We first constructed a vector plasmid with the green fluorescent protein (GFP) gene fused with the silkworm cytoplasmic actin gene (A3) promoter, and a helper plasmid with the Minos transposase gene controlled by the same A3 promoter. Injection of the vector and helper plasmid DNA into silkworm eggs produced transgenic animals in the following generation. The efficiency of transgenic silkworm production using this method was much lower than that obtained using piggyBac-mediated germ line transformation. However, >40-fold increase in the efficiency of producing transgenic silkworms was obtained using an in vitro synthesized source of Minos transposase mRNA. We conclude that the Minos transposon is a useful vector for construction of transgenic silkworms, particularly when in vitro synthesized mRNA is used. This is the first report showing that Minos can be used as a vector for germ-line transformation in lepidopteran insects.     

Application of the Saccharomyces cerevisiae FLP/FRT Recombination System in Filamentous Fungi for Marker Recycling and Construction of Knockout Strains Devoid of Heterologous Genes

To overcome the limited availability of antibiotic resistance markers in filamentous fungi, we adapted the FLP/FRT recombination system from the yeast Saccharomyces cerevisiae for marker recycling. We tested this system in the penicillin producer Penicillium chrysogenum using different experimental approaches. In a two-step application, we first integrated ectopically a nourseothricin resistance cassette flanked by the FRT sequences in direct repeat orientation (FRT-nat1 cassette) into a P. chrysogenum recipient. In the second step, the gene for the native yeast FLP recombinase, and in parallel, a codon-optimized P. chrysogenum flp (Pcflp) recombinase gene, were transferred into the P. chrysogenum strain carrying the FRT-nat1 cassette. The corresponding transformants were analyzed by PCR, growth tests, and sequencing to verify successful recombination events. Our analysis of several single- and multicopy transformants showed that only when the codon-optimized recombinase was present could a fully functional recombination system be generated in P. chrysogenum. As a proof of application of this system, we constructed a ΔPcku70 knockout strain devoid of any heterologous genes. To further improve the FLP/FRT system, we produced a flipper cassette carrying the FRT sites as well as the Pcflp gene together with a resistance marker. This cassette allows the controlled expression of the recombinase gene for one-step marker excision. Moreover, the applicability of the optimized FLP/FRT recombination system in other fungi was further demonstrated by marker recycling in the ascomycete Sordaria macrospora. Here, we discuss the application of the optimized FLP/FRT recombination system as a molecular tool for the genetic manipulation of filamentous fungi.Site-specific recombination is an important molecular tool for functional genetic studies in both prokaryotes and eukaryotes and is mediated by two major recombinase families, the resolvase/invertase family and the integrase family (9, 65). The resolvase/invertase family is characterized by the conserved catalytic amino acid serine, which allows intramolecular reactions, whereas the integrase family is able to mediate both intra- and intermolecular recombinations due to an autocatalytic activity of the conserved residue tyrosine (19, 35). The best-studied members of the integrase family are the λ integrase of bacteriophage λ, the recombinase Cre of Escherichia coli bacteriophage P1 (1, 62), the XerCD proteins of E. coli (6), and the eukaryotic FLP recombinase of the yeast Saccharomyces cerevisiae (9, 51). Common to all these systems are two different or identical recognition sites that serve as the DNA substrate for the recombinase.In recent years, recombinases have become important tools to manipulate genetically prokaryotes as well as eukaryotes. In this study, we have modified the FLP/FRT recombination system from yeast for application in filamentous fungi. The FLP/FRT recombination system is encoded by the 2μm (6.4 kb) plasmid that is present in most isolates of S. cerevisiae. Due to its stable partitioning and amplification system, this plasmid is available with a copy number of 60 to 100 copies per cell (16, 63). If the copy number drops below a certain level, the 45-kDa FLP recombinase catalyzes recombination between two 599-bp sites. These sequences are present in inverted orientation and allow the 2μm plasmid to exist in two conformations, namely, the A conformation when it is silent and the B conformation when it can replicate itself and thus maintain a constant plasmid copy number per cell (64). Analysis of the 599-bp inverted repeat sequence revealed that only 34 bp, the FLP recognition targets (FRT), are necessary for successful recombination (54). For every FLP-mediated recombination, a total of four FLP recombinases and two FRT sequences are needed. Two of the four proteins bind to one FRT sequence because every FRT has two 13-bp FLP-binding sites which are interrupted by an 8-bp spacer region. In this spacer DNA strand breakage takes place, producing 8-bp overhanging ends. After strand breakage, the overhanging ends of the two FRT fragments come together by complementary base pairing so that a recombinant FRT sequence is generated (2, 51).The FLP/FRT recombination system has been successfully applied in model organisms such as E. coli (10, 55) as well as in pathogenic and nonpathogenic Gram-negative and Gram-positive bacteria (53). In addition to prokaryotes, the FLP/FRT recombination system has been adapted for use in several eukaryotes (26). For example, it is well established in diverse yeasts (11, 41, 57, 60) and has been used successfully to modify the genomes of higher eukaryotes, including animal and human cell lines (5, 37, 58). Despite this tool''s application in higher eukaryotes, it has not yet been used in filamentous fungi. We present the first application of the FLP/FRT recombination system in a filamentous fungus, the major penicillin producer Penicillium chrysogenum. A limiting factor to manipulate P. chrysogenum genetically is the small number of suitable resistance markers. To overcome this problem, we generated a codon-optimized FLP/FRT recombination system to establish an efficient marker recycling system in this fungus. Using the optimized FLP/FRT recombination system, we demonstrate in a two-step approach the functionality of this system by construction of a marker-free P. chrysogenum strain lacking the ku70 (Pcku70) gene for optimized homologous recombination. A further advancement was the construction of a novel inducible nat1 flipper cassette allowing marker recycling in a one-step approach. The broader applicability of this recombination system was finally shown by its use in the model ascomycete Sordaria macrospora (34).     

<Emphasis Type="Italic">RAD52</Emphasis>-dependent and -independent homologous recombination initiated by Flp recombinase at a single <Emphasis Type="Italic">FRT</Emphasis> site flanked by direct repeats

We have devised a system for isolating yeast DNA sequences that are able to act as initiators of recombination leading to deletions in mitotically growing yeast cells. This system has allowed us to identify the FRT site of the 2μ site-specific recombinase Flp as such a sequence. We show that Flp is able to initiate recombination leading to deletions at a single FRT site in cir ^o strains. These results indicate that Flp is able to cleave a single FRT site, supporting the observation that the mechanism of cleavage by Flp is trans-horizontal. Interestingly, Flp can induce homologous recombination in both a RAD52-dependent and RAD52-independent manner. Our work provides a new system for the study of homologous recombination leading to deletions, in which the initiation step can be efficiently controlled. We discuss the possibility that Flp-induced, RAD52-independent events occur by single-strand annealing.     

Candida amazonensis FLP/FRT基因敲除系统的构建及初步验证

【目的】构建一个适用于Candida amazonensis抗性标记可重复使用的FLP/FRT基因敲除系统,并通过敲除C.amazonensis的丙酮酸脱羧酶基因(Pyruvate decarboxylase,PDC)对该系统进行初步验证。【方法】以gfpm(绿色荧光蛋白基因)为报告基因,通过添加相应诱导剂评估Spathaspora passalidarum来源启动子(SpXYLp、SpMAL6p、SpMAL1p、SpGAL1p)和Saccharomyces cerevisiae来源Sc GAL1p启动子在C.amazonensis中的诱导调控性能。选择严格诱导型启动子调控FLP重组酶的表达,并在FLP表达盒和潮霉素(Hygromycin B)抗性标记基因(hphm)两端添加同向重复的FRT位点,以PDC基因作为靶基因构建敲除盒PRFg HRP,转化宿主菌C.amazonensis CBS 12363,筛选得到阳性转化子后,通过添加诱导剂,表达FLP重组酶,实现FRT位点间片段切除。【结果】诱导调控实验表明启动子SpGAL1p(受半乳糖诱导)和SpMAL1p(受麦芽糖诱导)是适用于C.amazonensis的严格诱导型启动子。以SpGAL1p调控FLP基因表达,构建的敲除盒PRFg HRP成功转化宿主菌,获得阳性转化子C.amazonensis PDC01,通过添加半乳糖诱导,成功切除基因组中FLP表达盒和抗性标记盒,获得突变株C.amazonensis PDC02。【结论】首次建立了一个适用于C.amazonensis抗性标记可重复使用的FLP/FRT基因敲除系统,并利用该系统成功敲除了C.amazonensis内的PDC基因,为进一步利用代谢工程改造C.amazonensis酵母奠定了良好基础。     

Effects of transgenic overexpression of diapause hormone and diapause hormone receptor genes on non-diapause silkworm

Diapause is a state of developmental arrest that is most often observed in arthropods, especially insects. The domesticated silkworm, Bombyx mori, is a typical insect that enters diapause at an early embryonic stage. Previous studies have revealed that the diapause hormone (DH) signaling molecules, especially the core members DH and DH receptor 1 (DHR1), are crucial for the determination of embryonic diapause in diapause silkworm strains. However, whether they function in non-diapause silkworm strains remains largely unknown. Here, we generated two transgenic lines overexpressing DH or DHR1 genes in a non-diapause silkworm strain, Nistari. Our results showed that developmental expression patterns of DH and DHR1 are quite similar in transgenic silkworms: both genes are highly expressed in the mid to late stages of pupae and are most highly expressed in day-6 pupae but are expressed at very low levels in other developmental stages. Moreover, the overexpression of DH or DHR1 can affect the expression of diapause-related genes but is not sufficient to induce embryonic diapause in their offspring. This study provides new insights into the function of DH and DHR1 in a non-diapause silkworm strain.     

转蜘蛛拖牵丝蛋白基因家蚕蚕丝氨基酸组成及其机械性能

将以绿色荧光蛋白基因为报告基因、含有人工合成的1.6 kb的蜘蛛拖牵丝蛋白基因及转座子pig-gyBac的转基因载体成功导入减秋无滞育家蚕受精卵,得到转蜘蛛拖牵丝蛋白基因家蚕及绿色荧光茧。对转基因家蚕与对照家蚕丝素蛋白进行了氨基酸组成分析,结果表明转基因蚕茧丝素蛋白甘氨酸和丙氨酸的百分含量分别增加了1.65%和1.80%(平均值);对其生丝的机械性能进行了测试研究,结果表明转基因蚕茧生丝的伸长率降低,断裂强度和初始模量增加,且差异均显著,与理论预期结果吻合。结果表明转基因家蚕蚕丝的机械性能一定程度上得到了提高。     

A multifunctional reporter mouse line for Cre‐ and FLP‐dependent lineage analysis

The Cre/lox and FLP/FRT recombination systems have been used extensively for both conditional knockout and cell lineage analysis in mice. Here we report a new multifunctional Cre/FLP dual reporter allele (R26^NZG) that exhibits strong and apparently ubiquitous marker expression in embryos and adults. The reporter construct, which is driven by the CAG promoter, was knocked into the ROSA26 locus providing an open chromatin domain for consistent expression and avoiding site‐of‐integration effects often observed with transgenic reporters. R26^NZG directs Cre‐dependent nuclear‐localized β‐galactosidase (β‐gal) expression, and can be converted into a Cre‐dependent EGFP reporter (R26^NG) by germline excision of the FRT‐flanked nlslacZ cassette. Alternatively, germline excision of the floxed PGKNEO cassette in R26^NZG generates an FLP‐dependent EGFP reporter (R26^ZG) that expresses β‐gal in FLP‐nonexpressing cells. Finally, by the simultaneous use of both Cre and FLP deleters, R26^NZG allows lineage relationships to be interrogated with greater refinement than is possible with single recombinase reporter systems. genesis 47:107–114, 2009. © 2009 Wiley‐Liss, Inc.