FRT Sequences Research Articles

CRISPR/Cas9 and FLP-FRT mediated regulatory dissection of the BX-C of Drosophila melanogaster.

The homeotic genes or Hox define the anterior-posterior (AP) body axis formation in bilaterians and are often present on the chromosome in an order collinear to their function across the AP axis. However, there are many cases wherein the Hox are not collinear, but their expression pattern is conserved across the AP axis. The expression pattern of Hox is attributed to the cis-regulatory modules (CRMs) consisting of enhancers, initiators, or repressor elements that regulate the genes in a segment-specific manner. In the Drosophila melanogaster Hox complex, the bithorax complex (BX-C) and even the CRMs are organized in an order that is collinear to their function in the thoracic and abdominal segments. In the present study, the regulatorily inert regions were targeted using CRISPR/Cas9 to generate a series of transgenic lines with the insertion of FRT sequences. These FRT lines are repurposed to shuffle the CRMs associated with Abd-B to generate modular deletion, duplication, or inversion of multiple CRMs. The rearrangements yielded entirely novel phenotypes in the fly suggesting the requirement of such complex manipulations to address the significance of higher order arrangement of the CRMs. The functional map and the transgenic flies generated in this study are important resources to decipher the collective ability of multiple regulatory elements in the eukaryotic genome to function as complex modules.

CHOK1SV GS-KO SSI expression system: A combination of the Fer1L4 locus and glutamine synthetase selection.

There are an ever-increasing number of biopharmaceutical candidates in clinical trials fueling an urgent need to streamline the cell line development process. A critical part of the process is the methodology used to generate and screen candidate cell lines compatible with GMP manufacturing processes. The relatively large amount of clone phenotypic variation observed from conventional "random integration" (RI)-based cell line construction is thought to be the result of a combination of the position variegation effect, genome plasticity and clonal variation. Site-specific integration (SSI) has been used by several groups to temper the influence of the position variegation effect and thus reduce variability in expression of biopharmaceutical candidates. Following on from our previous reports on the application of the Fer1L4 locus for SSI in CHOK1SV (10E9), we have combined this locus and a CHOK1SV glutamine synthetase knockout (GS-KO) host to create an improved expression system. The host, CHOK1SV GS-KO SSI (HD7876), was created by homology directed integration of a targetable landing pad flanked with incompatible Frt sequences in the Fer1L4 gene. The targeting vector contains a promoterless GS expression cassette and monoclonal antibody (mAb) expression cassettes, flanked by Frt sites compatible with equivalent sites flanking the landing pad in the host cell line. SSI clones expressing four antibody candidates, selected in a streamlined cell line development process, have mAb titers which rival RI (1.0-4.5g/L) and robust expression stability (100% of clones stable through the 50 generation "manufacturing window" which supports commercial manufacturing at 12,000 L bioreactor scale).

Quick validation of genetic quality for conditional alleles in mice.

Site‐specific conditional inactivation technologies using Cre‐loxP or Flp‐FRT systems are becoming increasingly important for the elucidation of gene function and disease mechanism in vivo. A large number of gene knockout mouse models carrying complex conditional alleles have been generated by global community efforts and made available for biomedical researchers. The structures of conditional alleles in these mice are becoming increasingly complex and sophisticated, and so the validation of the genetic quality of these alleles is likewise becoming a laborious task for individual researchers. To ensure the reproducibility of conditional experiments, the researcher should confirm that loxP or FRT is integrated at the correct positions in the genome prior to start of the experiments. We report the successful design of universal PCR primers specific to loxP and FRT for the quick validation of conditional floxed and Flrted alleles. The primer set consists of forward and reverse primers complimentary to the loxP or FRT sequences with partial modifications at the 5ʹ end containing 6‐base restriction endonuclease recognition sites. The universal primer set was tested to detect genomic intervals between a pair of cis‐integrated loxP or FRT and was useful for quickly validating various floxed or Flrted alleles in conditional mice.

A novel Bxb1 integrase RMCE system for high fidelity site-specific integration of mAb expression cassette in CHO Cells.

As CHO cell line development for biotherapeutic production becomes more sophisticated through the availability of the CHO genome sequence, the ability to accurately and reproducibly engineer the host cell genome has become increasingly important. Multiple well characterized systems for site-specific integration will enable more complex cell line engineering to generate cell lines with desirable attributes. We built and characterized a novel recombinase mediated cassette exchange (RMCE) system using Bxb1 integrase and compared it to the commonly used Flp/FRT RMCE system. We first integrated a DNA construct flanked by either Bxb1 attachment sites or FRT sequences (referred to as a landing pad) into the Fer1L4 genomic locus of CHO-S cells using CRISPR/Cas9 mediated homologous recombination. We characterized the resulting clones harboring either the Bxb1 or Flp/FRT landing pad using whole genome resequencing to compare their genomes with the parental host cell line. We determined that each landing pad was specifically integrated into the Fer1L4 locus in the selected clones and observed no major structural changes in the genome or variations in copy number as a result of CRISPR/Cas9 modification. We subsequently tested the ability of the Bxb1 and Flp/FRT landing pad clones to perform proper RMCE with donor vectors containing identical mAb expression cassettes flanked by either Bxb1 attachment sites or FRT sites. We demonstrated that both RMCE systems were able to generate stable pools in a similar time frame with comparable mAb expression. Through genetic characterization of up to 24 clones derived from either system, we determined that the BxB1 RMCE system yielded higher fidelity RMCE events than the Flp/FRT system as evidenced by a higher percentage of clones with expected integration of the mAb cassette into the landing pad in the respective cell lines. We conclude that Bxb1 RMCE is an excellent alternative to Flp/FRT RMCE and valuable addition to our toolbox enabling the engineering of more sophisticated cell lines for biotherapeutic production. Biotechnol. Bioeng. 2017;114: 1837-1846. © 2017 Wiley Periodicals, Inc.

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the <em>AAVS1</em> Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells (hPSCs) is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming.

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the <em>AAVS1</em> Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells (hPSCs) is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming.

Construction of a restriction-less, marker-less mutant useful for functional genomic and metabolic engineering of the biofuel producer Clostridium acetobutylicum.

BackgroundClostridium acetobutylicum is a gram-positive, spore-forming, anaerobic bacterium capable of converting various sugars and polysaccharides into solvents (acetone, butanol, and ethanol). The sequencing of its genome has prompted new approaches to genetic analysis, functional genomics, and metabolic engineering to develop industrial strains for the production of biofuels and bulk chemicals.ResultsThe method used in this paper to knock-out or knock-in genes in C. acetobutylicum combines the use of an antibiotic-resistance gene for the deletion or replacement of the target gene, the subsequent elimination of the antibiotic-resistance gene with the flippase recombinase system from Saccharomyces cerevisiae, and a C. acetobutylicum strain that lacks upp, which encodes uracil phosphoribosyl-transferase, for subsequent use as a counter-selectable marker. A replicative vector containing (1) a pIMP13 origin of replication from Bacillus subtilis that is functional in Clostridia, (2) a replacement cassette consisting of an antibiotic resistance gene (MLSR) flanked by two FRT sequences, and (3) two sequences homologous to selected regions around target DNA sequence was first constructed. This vector was successfully used to consecutively delete the Cac824I restriction endonuclease encoding gene (CA_C1502) and the upp gene (CA_C2879) in the C. acetobutylicum ATCC824 chromosome. The resulting C. acetobutylicum Δcac1502Δupp strain is marker-less, readily transformable without any previous plasmid methylation and can serve as the host for the “marker-less” genetic exchange system. The third gene, CA_C3535, shown in this study to encode for a type II restriction enzyme (Cac824II) that recognizes the CTGAAG sequence, was deleted using an upp/5-FU counter-selection strategy to improve the efficiency of the method. The restriction-less marker-less strain and the method was successfully used to delete two genes (ctfAB) on the pSOL1 megaplasmid and one gene (ldhA) on the chromosome to get strains no longer producing acetone or l-lactate.ConclusionsThe restriction-less, marker-less strain described in this study, as well as the maker-less genetic exchange coupled with positive selection, will be useful for functional genomic studies and for the development of industrial strains for the production of biofuels and bulk chemicals.

Engineering Human Stem Cell Lines with Inducible Gene Knockout using CRISPR/Cas9

Precise temporal control of gene expression or deletion is critical for elucidating gene function in biological systems. However, the establishment of human pluripotent stem cell (hPSC) lines with inducible gene knockout (iKO) remains challenging. We explored building iKO hPSC lines by combining CRISPR/Cas9-mediated genome editing with the Flp/FRT and Cre/LoxP system. We found that "dual-sgRNA targeting" is essential for biallelic knockin of FRT sequences to flank the exon. We further developed a strategy to simultaneously insert an activity-controllable recombinase-expressing cassette and remove the drug-resistance gene, thus speeding up the generation of iKO hPSC lines. This two-step strategy was used to establish human embryonic stem cell (hESC) and induced pluripotent stem cell (iPSC) lines with iKO of SOX2, PAX6, OTX2, and AGO2, genes that exhibit diverse structural layout and temporal expression patterns. The availability of iKO hPSC lines will substantially transform the way we examine gene function in human cells.

Abstract IA25: Targeting K-Ras signaling in pancreatic cancer

Abstract K-RAS oncogenes have been implicated in about one fifth of all human cancers including pancreatic ductal adenocarcinoma (PDAC). We have developed genetically engineered mouse (GEM) models that closely recapitulate the natural history of this human neoplasia and use them to validate targets of potential therapeutic value. Briefly, we have crossed a GEM strain that harbors an endogenous K-RasG12V oncogene whose expression is controlled by a doxycycline-regulated Elastase promoter (via a Cre-lox system) with mice that carry conditional knock out mutations in loci encoding potential therapeutic targets. These targets are ablated by genetic means (also via the Cre-lox system) at the time of tumor initiation. Then, we follow the fate of the tumor in the absence of the target. This genetic-based strategy has also been used to interrogate the role of the inflammatory pathways by crossing this K-Ras driven GEM model with strains of mice defective for T and B lymphocytes (Rag-1 KO), IL6 (IL6 KO), Cox-2 (Cox-2 KO) and the NF-kB pathway (Ikkbeta KOlox mice). We have observed that ablation of any of these inflammatory mediators results in various degrees of tumor delay, but none of them had a significant impact on overall survival. These observations suggest that whereas blocking inflammation has an inhibitory effect on tumor development, its ultimate therapeutic utility to treat the disease is likely to be relatively minor. We have obtained more promising results ablating signaling components directly involved in K-Ras oncogenic signaling. For instance, we have observed that when PDAC tumors are initiated by expression of the resident K-RasG12V oncogene, ablation of its downstream c-Raf kinase completely prevents tumor development. Similar results were obtained when we knocked out the locus encoding the EGF receptor, a surprising result considering that the EGF receptor signals upstream of K-Ras. However, when the PDAC tumors were initiated by expressing the K-Ras oncogene along with inactivation of the p53 tumor suppressor, a common mutation in human PDAC, mice developed tumors in the absence of either c-Raf or the EGF receptor. However, mice displayed a significant increased (as much as 90%) in survival time. More recently, we have generated mice carrying both conditional c-Raf and EGF receptor alleles. These mice (n=14) have not developed even preneoplastic PanIN lesions at one year of age even in the absence of the p53 tumor suppressor. These observations indicate that EGF receptor signals through a different pathway that synergizes with the main Raf/Mek/Erk signaling cascade. We are now exploring whether concomitant ablation of c-Raf and EGF receptors will also induce a significant therapeutic effect in already developed K-Ras driven PDAC tumors lacking the p53 tumor suppressor. To this end, we have generated a new GEM model in which the resident K-Ras oncogene and the p53 tumor suppressor are flanked by frt sequences (instead of lox) and the doxycycline-regulated Elastase promoter directs the expression of the FLpase recombinase (instead of Cre). This compound GEM model allows us to separate tumor development (based on a frt/FLpase system) from ablation of the therapeutic targets (mediated by the classical cre-lox system). We hope that these genetic approaches will define therapeutic strategies that may eradicate aggressive K-Ras driven PDAC tumors with the ultimate goal to translate these results to the clinic. Citation Format: Mariano Barbacid. Targeting K-Ras signaling in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr IA25.

A genetic toolkit for tagging intronic MiMIC containing genes.

Previously, we described a large collection of Minos-Mediated Integration Cassettes (MiMICs) that contain two phiC31 recombinase target sites and allow the generation of a new exon that encodes a protein tag when the MiMIC is inserted in a codon intron (Nagarkar-Jaiswal et al., 2015). These modified genes permit numerous applications including assessment of protein expression pattern, identification of protein interaction partners by immunoprecipitation followed by mass spec, and reversible removal of the tagged protein in any tissue. At present, these conversions remain time and labor-intensive as they require embryos to be injected with plasmid DNA containing the exon tag. In this study, we describe a simple and reliable genetic strategy to tag genes/proteins that contain MiMIC insertions using an integrated exon encoding GFP flanked by FRT sequences. We document the efficiency and tag 60 mostly uncharacterized genes.

Healing of massive segmental femoral bone defects in minipigs by allogenic ASCs engineered with FLPo/Frt-based baculovirus vectors

Adipose-derived stem cells (ASCs) hold promise for bone regeneration but possess inferior osteogenesis potential. Allotransplantation of ASCs engineered with the BMP2/VEGF-expressing baculoviruses into rabbits healed critical-size segmental bone defects. To translate the technology to clinical applications, we aimed to demonstrate massive bone healing in minipigs that more closely mimicked the clinical scenarios, using a new hybrid baculovirus system consisting of BacFLPo expressing the codon-optimized FLP recombinase (FLPo) and the substrate baculovirus harboring the transgene flanked by Frt sequences. Co-transduction of minipig ASCs (pASCs) with BacFLPo and the substrate baculovirus enabled transgene cassette excision, recombination and minicircle formation in ≈73.7% of pASCs, which substantially prolonged the transgene (BMP2 and VEGF) expression to 28 days. When encoding BMP2, the FLPo/Frt-based system augmented the pASCs osteogenesis. Allotransplantation of the BMP2/VEGF-expressing pASCs into minipigs healed massive segmental bone defects (30 mm in length) at the mid-diaphysis of femora, as evaluated by computed tomography, positron emission tomography, histology, immunohistochemical staining and biochemical testing. The defect size was ≈15% of femoral length in minipigs and was equivalent to ≈60–70 mm of femoral defect in humans, thus the healing using pASCs engineered with the FLPo/Frt-based baculovirus represented a remarkable advance for the treatment of massive bone defects.

Ventricular septal defects in mice targeted deletion of versican in vascular smooth muscle cells

Purpose: To determine the function of versican (Vcan) in the cardiovascular system in vivo, we generated the versican conditional knockout mice, in which the Vcan gene was selectively ablated in vascular smooth muscle cells (SMCs). Versican is a large secreted chondroitin sulfate proteoglycan, which is important for cardiac development, and is also implicated in adult cardiovascular disease. Four splice variants of versican (V0, V1, V2, V3) have been identified, each containing two invariant domains, an N-terminal G1 domain that interact with hyaluronan, and a C-terminal G3 domain that interact with other extracellular matrix (ECM) molecules. As it has been reported that versican knockout mice died of cardiac defects at E10.5, the role of versican in cardiac development and disease has not been completely elucidated. Methods: SM22-Cre+:Vcanflox/flox conditional knockout mice were generated by combining Cre/loxP with a Flp/FRT system. In brief, a targeting vector harboring the Vcan flox allele was constructed by franking exon2 including start codon with loxP sites in the combination of a PGK-neoR cassette flanked by the FRT sequence. After obtaining Vcanflox/flox mice, they crossbred with SM22-Cre Tg mice, and the Vcan gene was selectively ablated in SMCs. The expression of different cardiac markers and ECM molecules were analyzed using histological and biochemical methods. Results: SM22-Cre+:Vcanflox/flox mice were viable and fertile, and they can reach their lifespan. The hearts in SM22-Cre+:Vcanflox/flox newborn mice showed normal cardiac looping; however, exhibited dilated heart and ventricular septal defects (VSD). The expressions of the a-actinin in their ventricular wall, which is a marker of cardiac muscle cell, decreased compared to wild type. Furthermore, though their arterial walls were thinner than wild type, elastic lamina was formed. Conclusions: Previously we generated the versican mutant mice (VcanΔ3/Δ3) whose versican lacked the A-subdomain of the G1 domain. The VcanΔ3/Δ3 mice died at neonatal period day 0, exhibiting the dilated heart and VSD. In this study, the targeted deletion of versican in SMCs leads to moderate cardiovascular defects with the dilated heart and VSD. SMCs transduce a variety of signals from a highly structured ECM that modulate cellular differentiation, migration, and proliferation. Our results suggest that the versican expression from SMCs is important for the cardiovascular development, and this study can lead to the treatment for congenital cardiovascular disease.

Augmented healing of critical-size calvarial defects by baculovirus-engineered MSCs that persistently express growth factors

Repair of large calvarial bony defects remains clinically challenging because successful spontaneous calvarial re-ossification rarely occurs. Although bone marrow-derived mesenchymal stem cells (BMSCs) genetically engineered with baculovirus (BV) for transient expression of osteogenic/angiogenic factors hold promise for bone engineering, we hypothesized that calvarial bone healing necessitates prolonged growth factor expression. Therefore, we employed a hybrid BV vector system whereby one BV expressed FLP while the other harbored the BMP2 (or VEGF) cassette flanked by Frt sequences. Transduction of rabbit BMSCs with the FLP/Frt-based BV vector led to FLP-mediated episome formation, which not only extended the BMP2/VEGF expression beyond 28 days but augmented the BMSCs osteogenesis. After allotransplantation into rabbits, X-ray, PET/CT, μCT and histological analyses demonstrated that the sustained BMP2/VEGF expression remarkably ameliorated the angiogenesis and regeneration of critical-size (8 mm) calvarial defects, when compared with the group implanted with BMSCs transiently expressing BMP2/VEGF. The prolonged expression by BMSCs accelerated the bone remodeling and regenerated the bone through the natural intramembranous pathway, filling ≈83% of the area and ≈63% of the volume in 12 weeks. These data implicated the potential of the hybrid BV vector to engineer BMSCs for sustained BMP2/VEGF expression and the repair of critical-size calvarial defects.

P035 Adipocyte derived stem cells favour healing in a new minipig model of cutaneous radiation syndrome through reepithelialisation and neoangiogenesis

Adipose-derived stem cells (ASCs) hold promise for bone regeneration but possess inferior osteogenesis potential. Allotransplantation of ASCs engineered with the BMP2/VEGF-expressing baculoviruses into rabbits healed critical-size segmental bone defects. To translate the technology to clinical applications, we aimed to demonstrate massive bone healing in minipigs that more closely mimicked the clinical scenarios, using a new hybrid baculovirus system consisting of BacFLPo expressing the codon-optimized FLP recombinase (FLPo) and the substrate baculovirus harboring the transgene flanked by Frt sequences. Co-transduction of minipig ASCs (pASCs) with BacFLPo and the substrate baculovirus enabled transgene cassette excision, recombination and minicircle formation in ≈73.7% of pASCs, which substantially prolonged the transgene (BMP2 and VEGF) expression to 28 days. When encoding BMP2, the FLPo/Frt-based system augmented the pASCs osteogenesis. Allotransplantation of the BMP2/VEGF-expressing pASCs into minipigs healed massive segmental bone defects (30 mm in length) at the mid-diaphysis of femora, as evaluated by computed tomography, positron emission tomography, histology, immunohistochemical staining and biochemical testing. The defect size was ≈15% of femoral length in minipigs and was equivalent to ≈60–70 mm of femoral defect in humans, thus the healing using pASCs engineered with the FLPo/Frt-based baculovirus represented a remarkable advance for the treatment of massive bone defects.

Targeted transgenesis through pronuclear injection of improved vectors into in vitro fertilized eggs

We recently described a new transgenic technique termed pronuclear injection-based targeted transgenesis (PITT) (Ohtsuka et al. 2010), which enables targeted integration of transgenes into predetermined loci (e.g. Rosa26) by Cre-loxP-based recombination in fertilized eggs, leading to stable and reproducible transgene expression. In this study, two steps of PITT were modified. The donor vector was improved by introducing an FLPe expression cassette (Fig. 1a). Therefore, selfremoval of the extra sequence flanked by FRT sequences occurs without additional FLPe administration. In addition, the plasmid backbone of the donor vector was modified from pUC119-to pBR322based to reduce plasmid instability, which is sometimes associated with genomic DNA fragment cloning into a high-copy plasmid. Moreover, in vitro fertilized eggs were used in this study; in the previous study, fertilized eggs obtained by natural mating were used. In this study, fertilized eggs ([100) were obtained simultaneously using epididymal spermatozoa isolated from a single male carrying the targeted allele. Results are listed in Fig. 1b. Three independent donor vectors, pAWK, pAWV and pAXV, were microinjected with a Cre expression vector into in vitro fertilized eggs. The resulting PITT efficiency was 5/93 (5.4%; 95% CI, 1.8–12.1%), which is similar to that reported previously (4.3%; 95% CI, 2.7–6.5%), indicating the feasibility of this modified method and reproducibility of PITT. Regarding removal of the extra sequence, the recombinasemediated cassette exchange (RMCE) allele was successfully created in 60% of founder mice (3/5; indicated by *). Although the other two founder mice did not possess the RMCE allele, the extra sequences of these founders can be successfully removed by crossing them with an FLPe deleter mouse (Ohtsuka et al. 2010). Notably, all tested lines (5/5) exhibited successful germline transmission Electronic supplementary material The online version of this article (doi:10.1007/s11248-011-9505-y) contains supplementary material, which is available to authorized users.

Generation of stable cell lines by site-specific integration of transgenes into engineered Chinese hamster ovary strains using an FLP- FRT system

Random integration linking genomic amplification is widely used to generate desired cell lines for stable and high-level expressing recombinant proteins. But this technique is laborious, and the expression level is unpredictable due to position effects. After reading many reports on gene amplification, we hypothesized that there should be several loci in the genome of Chinese hamster ovary (CHO) cells that allow not only high-level, but also stable gene expression. Based on this hypothesis, we constructed a plasmid pMCEscan, which introduces a site-specific recombinase-recognition sequence, FRT, for gene targeting into those sites. Another targeting vector, pcDNA5/ FRT, has an FRT sequence fused to a promoterless hygromycin-resistance gene that can be expressed only when correct gene targeting occurs. Using the pMCEscan, which is a novel and stringent selection system used to create a few high protein-producing clones, we constructed engineered CHO strains that can be used for high-level production of foreign proteins by gene targeting. We selected 28 CHO strains that expressed a high-level of reporter genes and carried one copy of the pMCEscan in their chromosomes, and we treated these strains with methotrexate (MTX) to evaluate dihydrofolate reductase (DHFR)-mediated gene amplification. Nine clones showed high-level tissue plasminogen activator (tPA) production without amplification. We then targeted other genes (tPA, secreted alkaline phosphatase (SEAP), erythropoietin (EPO)) to test the basal expression ability of nine CHO strains. CHO strains 8-1 and 8-11 consistently expressed high basal levels of the recombinant genes. Using this cell-vector system, we obtained the tPA stable high producers by gene targeting and gene amplification. This system allows for rapid generation of recombinant proteins without cloning and greatly simplifies selection of cell lines for the production of potential therapeutic proteins.

Nucleosome positioning in the NPTII neomycin phosphotransferase gene on a yeast plasmid in the repressed and active states

A yeast plasmid was constructed to contain a hybrid GAL-CYC promoter, the NPTII neomycin phosphotransferase gene, and the FRT sequence between them. The CYC part of the GAL-CYC promoter harbored four upstream activating sequences (UASs) and two close TATA boxes. NPTII was efficiently expressed upon induction with galactose, conferring G418 resistance on yeast cells. Nucleosome positioning was studied in repressed and induced NPTII in transformed cells. A stable positioning of three nucleosomes was detected under repressive conditions (growth on glucose). Two nucleosomes were on the CYC part of the promoter, one including both of the TATA boxes. The third nucleosome overlapped the FRT sequence and the start of the NPTII coding region. Each of the three nucleosomes displayed multiple positions, suggesting their sliding along DNA. After induction of NPTII expression with galactose, a sliding of two nucleosomes was detected, exposing the TATA box and a long promoter segment. The 5′-distal nucleosome moved closer to the UASs, bringing them closer to the TATA box, which was assumed to facilitate the assembly of the preinitiation complex. The two nucleosomes slid independently of each other. The second nucleosome moved towards the FRT sequence and repositioned at its nucleosome positioning signal. Galactose-induced expression did not affect the nucleosome positioning in the coding region of NPTII. Unidirectional sliding and repositioning were detected without induction after deacetylase inhibition with trichostatin A. Basal NPTII expression was observed without activation of the GAL-CYC promoter and after a spatial uncoupling of the coding sequence and promoter via gene inversion and was probably driven by the FRT TATA-like element, which is in the region permanently exposed in vivo.

Role of H2-calponin in Regulating Macrophage Motility and Phagocytosis

The actin cytoskeleton plays a major role in cell motility that is essential for the function of phagocytes. Calponin is an actin-associated regulatory protein. Here we report the finding of significant levels of the h2 isoform of calponin in peripheral blood cells of myeloid lineage. To study the functional significance, h2-calponin gene (Cnn2) interrupted mice were constructed. Germ line transmission of the Cnn2-flox-neo allele was obtained in chimeras from two independent clones of targeted embryonic stem cells. The insertion of the neo(R) cassette into intron 2 of the Cnn2 gene resulted in a significant knockdown of h2-calponin expression. Removing the frt-flanked neo(R) cassette by FLP1 recombinase rescued the knockdown effect. Cre recombinase-induced deletion of the loxP-flanked exon 2 eliminated the expression of h2-calponin protein. H2-calponin-free mice showed reduced numbers of peripheral blood neutrophils and monocytes. H2-calponin-free macrophages demonstrated a higher rate of proliferation and faster migration than that of h2-calponin-positive cells, consistent with a faster diapedesis of peripheral monocytes and neutrophils. H2-calponin-free macrophages showed reduced spreading in adhesion culture together with decreased tropomyosin in the actin cytoskeleton. The lack of h2-calponin also significantly increased macrophage phagocytotic activity, suggesting a novel mechanism to regulate phagocyte functions.

Foxb1‐driven Cre expression in somites and the neuroepithelium of diencephalon, brainstem, and spinal cord

We have knocked-in Cre-IRES-EGFP in the Foxb1 locus by homologous recombination in embryonic stem cells. We removed the PGK-neo cassette (which was flanked by FRT sequences) by crossing with the FLPeR deleter mouse. The Foxb1(Cre) line showed Cre recombinase activity as well as EGFP fluorescence reproducing Foxb1 expression accurately. By crossing Foxb1(Cre) mice with the ROSA26R and Z/AP mouse reporter lines we have been able to trace the lineage of Foxb1-expressing cells. Early transient expression of Foxb1 in the paraxial mesoderm translates into labeling of the somites. In the central nervous system (CNS), the Foxb1 lineage includes the thalamus and mammillary body (hypothalamus), brainstem, and the ventral spinal cord and floor plate.

An efficient and targeted gene integration system for high-level antibody expression

Random integration linking genomic amplification has been used to generate desired cell lines for stable and high-level expressing recombinant antibodies. But this technique is laborious, and the expression level is unpredictable due to position effects. Here, we have constructed a cell-vector system for high-level antibody expression using an FRT/FLP strategy to overcome position effects. The key is to target the FRT sequence to chromosomal locations where there is a high rate of transcription and gene amplification, and the amplified genes can be maintained. To screen desired loci with high transcriptional activity and amplifiable capacity, dual weakened markers (selectable galactosidase and amplifiable dihydrofolate reductase, DHFR) and the FRT sequence were synchronously cloned into a plasmid. After transfection of a Chinese hamster ovary host cell line with this plasmid, we selected 20 candidate cell lines from 721 individual clones. An antibody gene-targeting vector carrying an FRT-fused hygromycin gene was constructed to target antibody genes into the chromosomal FRT site by FLP recombinase. Three out of 20 cell lines can be used as host cells for site-specific recombination. By using southern blot and fluorescence in situ hybridization (FISH), a candidate engineered cell line, number 37, was chosen. It contains a single FRT-tagged locus in its genome. FISH analysis indicated that the antibody genes were all located at the original FRT-tagged locus in the genome of the gene-targeted and gene-amplified cell lines. Three kinds of recombinant antibodies were successfully expressed in candidate cell line 37. The highest producers produced more than 200 μg/ml of the antibody in 6 days of continuous culture in a spinner flask.