Targeted genetics in Drosophila cell lines: Inserting single transgenes in vitro

A long-standing problem with analyzing transgene expression in tissue-culture cells is the variation caused by random integration of different copy numbers of transfected transgenes. In mammalian cells, single transgenes can be inserted by homologous recombination but this process is inefficient in Drosophila cells. To tackle this problem, our group, and the Cherbas group, used recombination-mediated cassette exchange (RMCE) to introduce single-copy transgenes into specific locations in the Drosophila genome. In both cases, ?C31 was used to catalyze recombination between its target sequences attP in the genome, and attB flanking the donor sequence. We generated cell lines de novo with a single attP-flanked cassette for recombination, whereas, Cherbas et al. introduced a single attP-flanked cassette into existing cell lines. In both approaches, a 2-drug selection scheme was used to select for cells with a single copy of the donor sequence inserted by RMCE and against cells with random integration of multiple copies. Here we describe the general advantages of using RMCE to introduce genes into fly cells, the different attributes of the 2 methods, and how future work could make use of other recombinases and CRISPR/Cas9 genome editing to further enable genetic manipulation of Drosophila cells in vitro.     

The transgeneticist's toolbox: novel methods for the targeted modification of eukaryotic genomes

Classical techniques for gene transfer into mammalian cells involve tedious screening procedures to identify transgenic clones or animals with the appropriate level and stability of expression or with the correct developmental patterns. These first generation technologies are clearly inadequate for complex genetic strategies by which gene regulation can be studied in its entire complexity. While site-specific insertions can principally be achieved by homologous recombination or by adapting the recombination apparatus from phages or yeast, these methods usually lack the required efficiency or they perturb expression patterns by the co-insertion of prokaryotic vector parts. Virtually all of these problems can be overcome by recombinase-mediated cassette exchange (RMCE) techniques which cleanly replace a resident cassette that is flanked by two hetero-specific recombination target sites for a second cassette with the analogous design, presented on a targeting vector. After illustrating the fundamentals of site-specific recombination by selected experiments, the authors (arranged in the chronological order of their contribution) will describe their efforts to develop RMCE into a method of wide applicability. Further developments that have been initiated utilizing the particular potential of the RMCE principle will be outlined.     

Stable recombinase-mediated cassette exchange in Arabidopsis using Agrobacterium tumefaciens

Site-specific integration is an attractive method for the improvement of current transformation technologies aimed at the production of stable transgenic plants. Here, we present a Cre-based targeting strategy in Arabidopsis (Arabidopsis thaliana) using recombinase-mediated cassette exchange (RMCE) of transferred DNA (T-DNA) delivered by Agrobacterium tumefaciens. The rationale for effective RMCE is the precise exchange of a genomic and a replacement cassette both flanked by two heterospecific lox sites that are incompatible with each other to prevent unwanted cassette deletion. We designed a strategy in which the coding region of a loxP/lox5171-flanked bialaphos resistance (bar) gene is exchanged for a loxP/lox5171-flanked T-DNA replacement cassette containing the neomycin phosphotransferase (nptII) coding region via loxP/loxP and lox5171/lox5171 directed recombination. The bar gene is driven by the strong 35S promoter, which is located outside the target cassette. This placement ensures preferential selection of RMCE events and not random integration events by expression of nptII from this same promoter. Using root transformation, during which Cre was provided on a cotransformed T-DNA, 50 kanamycin-resistant calli were selected. Forty-four percent contained a correctly exchanged cassette based on PCR analysis, indicating the stringency of the selection system. This was confirmed for the offspring of five analyzed events by Southern-blot analysis. In four of the five analyzed RMCE events, there were no additional T-DNA insertions or they easily segregated, resulting in high-efficiency single-copy RMCE events. Our approach enables simple and efficient selection of targeting events using the advantages of Agrobacterium-mediated transformation.     

Targeted Integration of Single-Copy Transgenes in Drosophila melanogaster Tissue-Culture Cells Using Recombination-Mediated Cassette Exchange

Transfection of transgenes into Drosophila cultured cells is a standard approach for studying gene function. However, the number of transgenes present in the cell following transient transfection or stable random integration varies, and the resulting differences in expression level affect interpretation. Here we developed a system for Drosophila cell lines that allows selection of cells with a single-copy transgene inserted at a specific genomic site using recombination-mediated cassette exchange (RMCE). We used the φC31 integrase and its target sites attP and attB for RMCE. Cell lines with an attP-flanked genomic cassette were transfected with donor plasmids containing a transgene of interest (UAS-x), a dihydrofolate reductase (UAS-DHFR) gene flanked by attB sequences, and a thymidine kinase (UAS-TK) gene in the plasmid backbone outside the attB sequences. In cells undergoing RMCE, UAS-x and UAS-DHFR were exchanged for the attP-flanked genomic cassette, and UAS-TK was excluded. These cells were selected using methotrexate, which requires DHFR expression, and ganciclovir, which causes death in cells expressing TK. Pure populations of cells with one copy of a stably integrated transgene were efficiently selected by cloning or mass culture in ∼6 weeks. Our results show that RMCE avoids the problems associated with current methods, where transgene number is not controlled, and facilitates the rapid generation of Drosophila cell lines in which expression from a single transgene can be studied.     

Recombinase mediated cassette exchange into genomic targets using an adenovirus vector

Recombinase mediated cassette exchange (RMCE) is a process in which site-specific recombinases exchange one gene cassette flanked by a pair of incompatible target sites for another cassette flanked by an identical pair of sites. Typically one cassette is present in the host genome, whereas the other gene cassette is introduced into the host cell by chemical or biological means. We show here that the frequency of cassette exchange is dependent on the relative and absolute quantities of the transgene cassette and the recombinase. We were able to successfully modify genomic targets not only by electroporation or chemically mediated gene transfer but also by using an adenovirus vector carrying both the transgene cassette to be inserted and the recombinase coding region. RMCE proceeds efficiently in cells in which the adenovirus vector is able to replicate. In contrast, insufficient quantities of the transgene cassette are produced in cells in which the virus cannot replicate. Additional transfection of the transgene cassette significantly enhances the RMCE frequency. This demonstrates that an RMCE system in the context of a viral vector allows the site directed insertion of a transgene into a defined genomic site.     

A collection of sequenced and mapped Ds transposon insertion sites in Arabidopsis thaliana

Insertional mutagenesis is a powerful tool for generating knockout mutations that facilitate associating biological functions with as yet uncharacterized open reading frames (ORFs) identified by genomic sequencing or represented in EST databases. We have generated a collection of Dissociation (Ds) transposon lines with insertions on all 5 Arabidopsis chromosomes. Here we report the insertion sites in 260 independent single-transposon lines, derived from four different Ds donor sites. We amplified and determined the genomic sequence flanking each transposon, then mapped its insertion site by identity of the flanking sequences to the corresponding sequence in the Arabidopsis genome database. This constitutes the largest collection of sequence-mapped Ds insertion sites unbiased by selection against the donor site. Insertion site clusters have been identified around three of the four donor sites on chromosomes 1 and 5, as well as near the nucleolus organizers on chromosomes 2 and 4. The distribution of insertions between ORFs and intergenic sequences is roughly proportional to the ratio of genic to intergenic sequence. Within ORFs, insertions cluster near the translational start codon, although we have not detected insertion site selectivity at the nucleotide sequence level. A searchable database of insertion site sequences for the 260 transposon insertion sites is available at http://sgio2.biotec.psu.edu/sr. This and other collections of Arabidopsis lines with sequence-identified transposon insertion sites are a valuable genetic resource for functional genomics studies because the transposon location is precisely known, the transposon can be remobilized to generate revertants, and the Ds insertion can be used to initiate further local mutagenesis.     

Targeted engineering of the Drosophila genome

The application of phiC31 phage integrase in Drosophila for unidirectional and site-specific DNA integration was pioneered by Groth et al. in 2004 1 and quickly triggered a wave of innovative tools taking advantage of these unique properties of phiC31. Three recent papers have further developed novel approaches that combine the phiC31-mediated DNA integration with the homologous recombination (HR)-based gene targeting 2 3 for the purpose of efficient and targeted modifications of Drosophila genomic loci. Despite significant differences, the general strategies are similar in principle in the SIRT (site-specific integrase mediated repeated targeting) approach by Gao et al. 4, the IMAGO (integrase-mediated approach for gene knock-out) approach by Choi et al. 5 and the genomic engineering approach developed by our group 6. All three use HR-based gene targeting to first implant a single or a pair of phiC31-attP recombination sites into the target locus. Flies carrying such targeted insertions of attP sites can then be used as "founder lines", in which modified DNA sequences ("knock-in DNA") can be repeatedly and efficiently inserted back into the target locus via phiC31-mediated integration. Thus, by carrying out the targeting experiments only once, one can then directedly and efficiently modify the target locus into virtually any desired knock-in allele. Here we give a brief overview of the SIRT, IMAGO, and genomic engineering approaches and propose a revised genomic engineering scheme in which a single ends-out targeting event will generate founder lines suitable for both recombinase-mediated cassette exchange (RMCE) and single-site based integration of knock-in DNA.     

A collection of sequenced and mapped Ds transposon insertion sites in Arabidopsis thaliana

Insertional mutagenesis is a powerful tool for generating knockout mutations that facilitate associating biological functions with as yet uncharacterized open reading frames (ORFs) identified by genomic sequencing or represented in EST databases. We have generated a collection of Dissociation(Ds) transposon lines with insertions on all 5 Arabidopsischromosomes. Here we report the insertion sites in 260 independent single-transposon lines, derived from four different Ds donor sites. We amplified and determined the genomic sequence flanking each transposon, then mapped its insertion site by identity of the flanking sequences to the corresponding sequence in the Arabidopsisgenome database. This constitutes the largest collection of sequence-mapped Ds insertion sites unbiased by selection against the donor site. Insertion site clusters have been identified around three of the four donor sites on chromosomes 1 and 5, as well as near the nucleolus organizers on chromosomes 2 and 4. The distribution of insertions between ORFs and intergenic sequences is roughly proportional to the ratio of genic to intergenic sequence. Within ORFs, insertions cluster near the translational start codon, although we have not detected insertion site selectivity at the nucleotide sequence level. A searchable database of insertion site sequences for the 260 transposon insertion sites is available at http://sgio2.biotec.psu.edu/sr. This and other collections of Arabidopsislines with sequence-identified transposon insertion sites are a valuable genetic resource for functional genomics studies because the transposon location is precisely known, the transposon can be remobilized to generate revertants, and the Ds insertion can be used to initiate further local mutagenesis.     

New transposons to generate GFP protein fusions in Candida albicans

Transposon elements are important tools for gene function analysis, for example they can be used to easily create genome-wide collections of insertion mutants. Transposons may also carry sequences coding for an epitope or fluorescent marker useful for protein expression and localization analysis. We have developed three new Tn5-based transposons that incorporate a GFP (green fluorescent protein) coding sequence to generate fusion proteins in the important fungal pathogen Candida albicans. Each transposon also contains the URA3 and Kan(R) genes for yeast and bacterial selection, respectively. After in vitro transposition, the insertional allele is transferred to the chromosomal locus by homologous recombination. Transposons Tn5-CaGFP and Tn5-CaGFP-URA3::FLIP can generate C-terminal truncated GFP fusions. A URA3 flipper recycling cassette was incorporated into the transposon Tn5-CaGFP-URA3::FLIP. After the induction of Flip recombinase to excise the marker, the heterozygous strain is transformed again in order to obtain a GFP-tagged homozygous strains. In the Tn5-CaGFP-FL transposon the markers are flanked by a rare-cutting enzyme. After in vitro transposition into a plasmid-borne target gene, the markers are eliminated by restriction digestion and religation, resulting in a construct coding for full-length GFP-fusion proteins. This transposon can generate plasmid libraries of GFP insertions in proteins where N- or C-terminal tagging may alter localization. We tested our transposon system by mutagenizing the essential septin CDC3 gene. The results indicate that the Cdc3 C-terminal extension is important for correct septin filament assembly. The transposons described here provide a new system to obtain global gene expression and protein localization data in C. albicans.     

Target site choice of the related transposable elements Tc1 and Tc3 of Caenorhabditis elegans.

We have investigated the target choice of the related transposable elements Tc1 and Tc3 of the nematode C. elegans. The exact locations of 204 independent Tc1 insertions and 166 Tc3 insertions in an 1 kbp region of the genome were determined. There was no phenotypic selection for the insertions. All insertions were into the sequence TA. Both elements have a strong preference for certain positions in the 1 kbp region. Hot sites for integration are not clustered or regularly spaced. The orientation of the integrated transposon has no effect on the distribution pattern. We tested several explanations for the target site preference. If simple structural features of the DNA (e.g. bends) would mark hot sites, we would expect the patterns of the two related transposons Tc1 and Tc3 to be similar; however we found them to be completely different. Furthermore we found that the sequence at the donor site has no effect on the choice of the new insertion site, because the insertion pattern of a transposon that jumps from a transgenic donor site is identical to the insertion pattern of transposons jumping from endogenous genomic donor sites. The most likely explanation for the target choice is therefore that the primary sequence of the target site is recognized by the transposase. However, alignment of the Tc1 and Tc3 integration sites does not reveal a strong consensus sequence for either transposon.     

Site-specific transformation of Drosophila via phiC31 integrase-mediated cassette exchange

Position effects can complicate transgene analyses. This is especially true when comparing transgenes that have inserted randomly into different genomic positions and are therefore subject to varying position effects. Here, we introduce a method for the precise targeting of transgenic constructs to predetermined genomic sites in Drosophila using the C31 integrase system in conjunction with recombinase-mediated cassette exchange (RMCE). We demonstrate the feasibility of this system using two donor cassettes, one carrying the yellow gene and the other carrying GFP. At all four genomic sites tested, we observed exchange of donor cassettes with an integrated target cassette carrying the mini-white gene. Furthermore, because RMCE-mediated integration of the donor cassette is necessarily accompanied by loss of the target cassette, we were able to identify integrants simply by the loss of mini-white eye color. Importantly, this feature of the technology will permit integration of unmarked constructs into Drosophila, even those lacking functional genes. Thus, C31 integrase-mediated RMCE should greatly facilitate transgene analysis as well as permit new experimental designs.     

Gene tagging and gene replacement using recombinase-mediated cassette exchange in Schizosaccharomyces pombe

Cre/lox site-specific recombination systems provide important tools for genetic manipulation. Here we present an efficient method for gene tagging and gene replacement using Cre recombinase-mediated cassette exchange (RMCE). The cassette consists of the S. pombe ura4(+) selectable marker flanked by a wild-type loxP site at one end and by a modified heterospecific lox site (loxM3) at the other. The cassette is stable because the flanking lox sites cannot recombine with each other. Following integration of the cassette at the chosen chromosomal locus, exchange is achieved by introducing a Cre-expression plasmid containing an equivalent cassette containing the required tag or gene sequence. Recombinants are selected by uracil prototrophy using the reagent 5-fluoroorotic acid (5-FOA). The cassette exchange system provides for repetitive integrations at the same locus, allowing different protein tags or gene sequences to be integrated quickly and efficiently. We have established a range of reagents and verified utility by C-terminally tagging the S. pombe rad4 and swi1 genes with yEGFP and the yEGFP derivatives yECFP and yECitrine and by transferring the coding sequence for both genes.     

Repeated integration of antibody genes into a pre-selected chromosomal locus of CHO cells using an accumulative site-specific gene integration system

We previously reported an accumulative site-specific gene integration system using Cre recombinase and mutated loxP sites, where a recombinase-mediated cassette exchange (RMCE) reaction is repeatable. This gene integration system was applied for antibody production using recombinant Chinese hamster ovary (CHO) cells. We introduced an exchange cassette flanked by wild-type and mutated loxP sites into the chromosome of CHO cells for the establishment of recipient founder cells. Then, the donor plasmids including an expression cassette for an antibody gene flanked by a compatible pair of loxP sites were prepared. The donor plasmid and a Cre expression vector were co-transfected into the founder CHO cells to give rise to RMCE in the CHO genome, resulting in site-specific integration of the antibody gene. The RMCE procedure was repeated to increase the copy numbers of the integrated gene. Southern blot and genomic PCR analyses for the established cells revealed that the transgenes were integrated into the target site. Antibody production determined by ELISA and western blotting was increased corresponding to the number of transgenes. These results indicate that the accumulative site-specific gene integration system could provide a useful tool for increasing the productivity of recombinant proteins.     

Remarkable site specificity of local transposition into the Hsp70 promoter of Drosophila melanogaster

Heat-shock genes have numerous features that ought to predispose them to insertional mutagenesis via transposition. To elucidate the evolvability of heat-shock genes via transposition, we have exploited a local transposition technique and Drosophila melanogaster strains with EPgy2 insertions near the Hsp70 gene cluster at 87A7 to produce numerous novel EPgy2 insertions into these Hsp70 genes. More than 50% of 45 independent insertions were made into two adjacent nucleotides in the proximal promoter at positions -96 and -97, and no insertions were into a coding or 3'-flanking sequence. All inserted transposons were in inverse orientation to the starting transposon. The frequent insertion into nucleotides -96 and -97 is consistent with the DNase hypersensitivity, absence of nucleosomes, flanking GAGA-factor-binding sites, and nucleotide sequence of this region. These experimental insertions recapitulated many of the phenotypes of natural transposition into Hsp70: reduced mRNA expression, less Hsp70 protein, and decreased inducible thermotolerance. The results suggest that the distinctive features of heat-shock promoters, which underlie the massive and rapid expression of heat-shock genes upon heat shock, also are a source of evolutionary variation on which natural selection can act.     

HK022 bacteriophage Integrase mediated RMCE as a potential tool for human gene therapy

HK022 coliphage site-specific recombinase Integrase (Int) can catalyze integrative site-specific recombination and recombinase-mediated cassette exchange (RMCE) reactions in mammalian cell cultures. Owing to the promiscuity of the 7 bp overlap sequence in its att sites, active ‘attB’ sites flanking human deleterious mutations were previously identified that may serve as substrates for RMCE reactions for future potential gene therapy. However, the wild type Int proved inefficient in catalyzing such RMCE reactions. To address this low efficiency, variants of Int were constructed and examined by integrative site-specific recombination and RMCE assays in human cells using native ‘attB’ sites. As a proof of concept, various Int derivatives have demonstrated successful RMCE reactions using a pair of native ‘attB’ sites that were inserted as a substrate into the human genome. Moreover, successful RMCE reactions were demonstrated in native locations of the human CTNS and DMD genes whose mutations are responsible for Cystinosis and Duchene Muscular Dystrophy diseases, respectively. This work provides a steppingstone for potential downstream therapeutic applications.     

The streptomyces genome contains multiple pseudo-attB sites for the (phi)C31-encoded site-specific recombination system

The integrase from the Streptomyces phage (phi)C31 is a member of the serine recombinase family of site-specific recombinases and is fundamentally different from that of lambda or its relatives. Moreover, (phi)C31 int/attP is used widely as an essential component of integration vectors (such as pSET152) employed in the genetic analysis of Streptomyces species. phiC31 or integrating plasmids containing int/attP have been shown previously to integrate at a locus, attB, in the chromosome. The DNA sequences of the attB sites of various Streptomyces species revealed nonconserved positions. In particular, the crossover site was narrowed to the sequence 5'TT present in both attP and attB. Strains of Streptomyces coelicolor and S. lividans were constructed with a deletion of the attB site ((Delta)attB), and pSET152 was introduced into these strains by conjugation. Thus, secondary or pseudo-attB sites were identified by Southern blotting and after rescue of plasmids containing DNA flanking the insertion sites from the chromosome. The sequences of the integration sites had similarity to those of attB. Analysis of the insertions of pSET152 into both attB(+) and (Delta)attB strains indicated that this plasmid can integrate at several loci via independent recombination events within a transconjugant.     

piggyBac转座子AgoPLE1.1在黑腹果蝇种系 转化中的应用

【目的】通过检测黑腹果蝇Drosophila melanogaster中piggyBac (PB)转座子AgoPLE1.1的转化活性,明确AgoPLE1.1开发为昆虫转基因载体的潜力。【方法】构建AgoPLE1.1转座酶辅助质粒pAgoHsp和带有红色荧光标记的供体质粒pXLAgo-PUbDsRed,辅助质粒和供体质粒以170 ng/μL∶400 ng/μL, 90 ng/μL∶200 ng/μL和90 ng/μL∶100 ng/μL 3种不同的比例混合后分别注射新鲜的W¹¹¹⁸ 黑腹果蝇胚胎,筛选注射后代中的转基因黑腹果蝇个体;利用Southern杂交验证转基因黑腹果蝇中AgoPLE1.1转座子的插入拷贝数;利用染色体步移技术克隆AgoPLE1.1插入位点旁侧序列,明确AgoPLE1.1转座子的转座特征。【结果】AgoPLE1.1转座子在黑腹果蝇中具有转化活性,转基因频率为1.32%~1.94%。Southern杂交结果显示,AgoPLE1.1转座子在黑腹果蝇中至少有6个插入位点。染色体步移法克隆了其中4个位点,分别位于黑腹果蝇的3R, 3L, 2L和X染色体,并且AgoPLE1.1转座子在黑腹果蝇染色体中的整合带有供体质粒的骨架。【结论】PB转座子AgoPLE1.1仅可以在黑腹果蝇中以较低的频率进行非精确的剪切和转座,不具有开发为新型昆虫转基因载体的潜力。