Targeted Gene Integration Research Articles

HisB as novel selection marker for gene targeting approaches in Aspergillus niger

BackgroundFor Aspergillus niger, a broad set of auxotrophic and dominant resistance markers is available. However, only few offer targeted modification of a gene of interest into or at a genomic locus of choice, which hampers functional genomics studies. We thus aimed to extend the available set by generating a histidine auxotrophic strain with a characterized hisB locus for targeted gene integration and deletion in A. niger.ResultsA histidine-auxotrophic strain was established via disruption of the A. niger hisB gene by using the counterselectable pyrG marker. After curing, a hisB-, pyrG- strain was obtained, which served as recipient strain for further studies. We show here that both hisB orthologs from A. nidulans and A. niger can be used to reestablish histidine prototrophy in this recipient strain. Whereas the hisB gene from A. nidulans was suitable for efficient gene targeting at different loci in A. niger, the hisB gene from A. niger allowed efficient integration of a Tet-on driven luciferase reporter construct at the endogenous non-functional hisB locus. Subsequent analysis of the luciferase activity revealed that the hisB locus is tight under non-inducing conditions and allows even higher luciferase expression levels compared to the pyrG integration locus.ConclusionTaken together, we provide here an alternative selection marker for A. niger, hisB, which allows efficient homologous integration rates as well as high expression levels which compare favorably to the well-established pyrG selection marker.

GeneHancer: genome-wide integration of enhancers and target genes in GeneCards.

A major challenge in understanding gene regulation is the unequivocal identification of enhancer elements and uncovering their connections to genes. We present GeneHancer, a novel database of human enhancers and their inferred target genes, in the framework of GeneCards. First, we integrated a total of 434 000 reported enhancers from four different genome-wide databases: the Encyclopedia of DNA Elements (ENCODE), the Ensembl regulatory build, the functional annotation of the mammalian genome (FANTOM) project and the VISTA Enhancer Browser. Employing an integration algorithm that aims to remove redundancy, GeneHancer portrays 285 000 integrated candidate enhancers (covering 12.4% of the genome), 94 000 of which are derived from more than one source, and each assigned an annotation-derived confidence score. GeneHancer subsequently links enhancers to genes, using: tissue co-expression correlation between genes and enhancer RNAs, as well as enhancer-targeted transcription factor genes; expression quantitative trait loci for variants within enhancers; and capture Hi-C, a promoter-specific genome conformation assay. The individual scores based on each of these four methods, along with gene–enhancer genomic distances, form the basis for GeneHancer’s combinatorial likelihood-based scores for enhancer–gene pairing. Finally, we define ‘elite’ enhancer–gene relations reflecting both a high-likelihood enhancer definition and a strong enhancer–gene association.GeneHancer predictions are fully integrated in the widely used GeneCards Suite, whereby candidate enhancers and their annotations are displayed on every relevant GeneCard. This assists in the mapping of non-coding variants to enhancers, and via the linked genes, forms a basis for variant–phenotype interpretation of whole-genome sequences in health and disease. Database URL: http://www.genecards.org/

Targeted integration of genes in Xenopus tropicalis.

With the successful establishment of both targeted gene disruption and integration methods in the true diploid frog Xenopus tropicalis, this excellent vertebrate genetic model now is making a unique contribution to modelling human diseases. Here, we summarize our efforts on establishing homologous recombination-mediated targeted integration in Xenopus tropicalis, the usefulness, and limitation of targeted integration via the homology-independent strategy, and future directions on how to further improve targeted gene integration in Xenopus tropicalis.

Long-Term Stable and Tightly Controlled Expression of Recombinant Proteins in Antibiotics-Free Conditions.

Plasmid-based gene expression is a fundamental tool in the field of biotechnology. However, overexpression of genes of interest with multi-copy plasmids often causes detrimental effects on host cells. To overcome this problem, chromosomal integration of target genes has been used for decades; however, insufficient protein expression occurred with this method. In this study, we developed a novel cloning and expression system named the chromosomal vector (ChroV) system, that has features of stable and high expression of target genes on the F′ plasmid in the Escherichia coli JM109(DE3) strain. We used an RMT cluster (KCTC 11994BP) containing a silent cat gene from a previous study to clone a gene into the F′ plasmid. The ChroV system was applied to clone two model targets, GFPuv and carotenoids gene clusters (4 kb), and successfully used to prove the inducible tightly regulated protein expression in the F′ plasmid. In addition, we verified that the expression of heterologous genes in ChroV system maintained stably in the absence of antibiotics for 1 week, indicating ChroV system is applicable to antibiotics-free production of valuable proteins. This protocol can be widely applied to recombinant protein expression for antibiotics-free, stable, and genome-based expression, providing a new platform for recombinant protein synthesis in E. coli. Overall, our approach can be widely used for the economical and industrial production of proteins in E. coli.

Novel method to rescue a lethal phenotype through integration of target gene onto the X-chromosome.

The loss-of-function mutations of serine protease inhibitor, Kazal type 1 (SPINK1) gene are associated with human chronic pancreatitis, but the underlying mechanisms remain unknown. We previously reported that mice lacking Spink3, the murine homologue of human SPINK1, die perinatally due to massive pancreatic acinar cell death, precluding investigation of the effects of SPINK1 deficiency. To circumvent perinatal lethality, we have developed a novel method to integrate human SPINK1 gene on the X chromosome using Cre-loxP technology and thus generated transgenic mice termed “X-SPINK1“. Consistent with the fact that one of the two X chromosomes is randomly inactivated, X-SPINK1 mice exhibit mosaic pattern of SPINK1 expression. Crossing of X-SPINK1 mice with Spink3+/− mice rescued perinatal lethality, but the resulting Spink3−/−;XXSPINK1 mice developed spontaneous pancreatitis characterized by chronic inflammation and fibrosis. The results show that mice lacking a gene essential for cell survival can be rescued by expressing this gene on the X chromosome. The Spink3−/−;XXSPINK1 mice, in which this method has been applied to partially restore SPINK1 function, present a novel genetic model of chronic pancreatitis.

High-level expression and characterization of a novel serine protease in Pichia pastoris by multi-copy integration.

A novel serine protease from Trichoderma koningii (SPTK) was synthesized and expressed in Pichia pastoris. The recombinant SPTK was completely inhibited by phenyl methyl sulfonyl fluoride (PMSF), suggesting that SPTK belonged to the subgroup of serine proteases. The optimum pH and temperature for the recombinant SPTK reaction were 6.0 and 55°C, respectively. SPTK performed a tolerance to most organic solvents and metal ions, and the addition of Triton X-100 exhibited an activation of SPTK up to 243% of its initial activity but SDS strongly inhibited. Moreover, our study showed that a portion of SPTK was N-glycosylated during fermentation. The activity and thermal stability of the recombinant SPTK were improved after the removal of glycosylation, and the N-glycosylation of SPTK could be efficiently removed through co-culture with P. pastoris strains expressing Endo-β-N-acetylglucosaminidase H. We constructed expression vectors harboring from one to four repeats of Sptk-expressing cassettes via an in vitro BioBrick assembly approach. And the result of quantitative polymerase chain reaction (qPCR) indicated that the tandem expression cassettes were integrated into the genome of P. pastoris through a single recombination event. These strains were used to study the correlation between the gene copy number and the expression level of SPTK. The results of qPCR and enzyme activity assays indicated that the copy number variation of Sptk gene generally had a positive effect on the expression level of SPTK, while an increase in integration of target gene did not guarantee its high expression. The maximum yield and specific activity of SPTK in P. pastoris were obtained from the recombinant yeast strain harboring two-copy tandem Sptk-expressing cassettes, the yield reached 0.48g/l after a 6-d induction using menthol in shake flasks and 3.2g/l in high-density fermentation with specific activity of 5200U/mg. In addition, the recombinant SPTK could efficiently degrade chicken feather and hydrolyzed the gelatin layer of photographic film. These properties made the recombinant SPTK a suitable candidate for industrial applications and for eliminating the pollution of keratin.

Targeted, homology-driven gene insertion in stem cells by ZFN-loaded 'all-in-one' lentiviral vectors.

Biased integration remains a key challenge for gene therapy based on lentiviral vector technologies. Engineering of next-generation lentiviral vectors targeting safe genomic harbors for insertion is therefore of high relevance. In a previous paper (Cai et al., 2014a), we showed the use of integrase-defective lentiviral vectors (IDLVs) as carriers of complete gene repair kits consisting of zinc-finger nuclease (ZFN) proteins and repair sequences, allowing gene correction by homologous recombination (HR). Here, we follow this strategy to engineer ZFN-loaded IDLVs that insert transgenes by a homology-driven mechanism into safe loci. This insertion mechanism is driven by time-restricted exposure of treated cells to ZFNs. We show targeted gene integration in human stem cells, including CD34(+) hematopoietic progenitors and induced pluripotent stem cells (iPSCs). Notably, targeted insertions are identified in 89% of transduced iPSCs. Our findings demonstrate the applicability of nuclease-loaded 'all-in-one' IDLVs for site-directed gene insertion in stem cell-based gene therapies.

Accelerated homology-directed targeted integration of transgenes in Chinese hamster ovary cells via CRISPR/Cas9 and fluorescent enrichment.

Targeted gene integration into site-specific loci can be achieved in Chinese hamster ovary (CHO) cells via CRISPR/Cas9 genome editing technology and the homology-directed repair (HDR) pathway. The low efficiency of HDR often requires antibiotic selection, which limits targeted integration of multiple genes at multiple sites. To improve HDR-mediated targeted integration, while avoiding the use of selection markers, chemical treatment for increased HDR, and fluorescent enrichment of genome-edited cells was assessed in CHO cells. Chemical treatment did not improve HDR-mediated targeted integration. In contrast, fluorescent markers in Cas9 and donor constructs enable FACS enrichment, resulting in a threefold increase in the number of cells with HDR-mediated genome editing. Combined with this enrichment method, large transgenes encoding model proteins (including an antibody) were successfully targeted integrated. This approach provides a simple and fast strategy for targeted generation of stable CHO production cell lines in a rational way. Biotechnol. Bioeng. 2016;113: 2518-2523. © 2016 Wiley Periodicals, Inc.

127. Lentiviral Protein Transduction for Tailored Genome Editing and Site-Directed Gene Insertion

Therapeutic use of site-directed endonucleases relies on safe and effective cellular delivery, preferentially resulting in short-term enzymatic activity. Based on the packaging of Gag/GagPol-fused heterologous proteins into VSV-G-pseudotyped lentivirus-derived particles, we have established lentiviral protein transduction for delivery of DNA transposases and custom-made endonucleases. Up to 24% of targeted CCR5 and AAVS1 alleles were disrupted in primary cells, including normal human dermal fibroblasts and primary keratinocytes, exposed to lentiviral particles loaded with zinc-finger nucleases (ZFNs). By exposing human 293 cells to ‘all-in-one’ integrase-defective lentiviral vectors (IDLVs) containing a complete gene repair kit consisting of ZFNs and viral RNA carrying the donor sequence for homology-directed repair, correction of genomic mutations was obtained in more than 8% of treated cells. As shown by confocal microscopy, ZFN proteins were abundant within transduced cells one hour after initial virus exposure, but were short-lived and gone after 24 hours. In accordance, under conditions supporting comparable CCR5 indel rates, disruption of the nearby CCR2 off-target site was reduced by lentiviral delivery of ZFNs targeting CCR5 relative to a conventional transfection-based approach. As biased and uncontrolled integration into genes remains a key challenge for gene therapies based on lentiviral vector technologies, we engineered ZFN-loaded IDLVs with the capacity to insert transgenes into the human CCR5 and AAVS1 loci by a homology-driven mechanism. Targeted gene integration into safe genomic loci was observed in human cell lines (85% of analyzed clones) and in human stem cells, including CD34+ hematopoietic progenitors and induced pluripotent stem cells (iPSCs). Notably, targeted transgene insertion into safe harbors was identified in all of 23 analyzed iPSC clones. Altogether, our findings generate a new platform for targeted genome engineering based on lentiviral delivery of complete gene repair or gene insertion kits.

315. Highly Efficient Targeted Addition of Large XIST Transgenes in a Population of Somatic Cells

Recent advances in genome editing technologies enable precise and corrective genome modification to treat genetic diseases in a targeted way. High efficiency of genome editing can be achieved through error-prone NHEJ pathway, however, the absolute rate of precise genome editing through homology-directed repair (HDR) pathway remains relatively low, especially for targeted addition of large DNA sequences. Although it is sufficient for the generation of cell lines derived from single targeted clones when coupled with drug selection, poor rates of homologous recombination greatly limit the practical application of targeted gene integration in somatic tissue. XIST encodes a large long non-coding RNA (19 kb) that plays an essential role in X chromosome inactivation in mammalian females. Recently, we have shown that XIST can be inserted to chromosome 21 and silence the extra copy of entire chromosome in Down syndrome stem cells derived from a single-targeted clones. Successful trisomy silencing in vitro holds promise for potential development of ‘chromosome therapy” in patients with Down syndrome. Here, using Zinc finger nuclease (ZFN) technology, we tested the feasibility of targeted addition of large XIST transgenes into different loci of human genome in a population of transformed cells, as well as primary fibroblasts. Our results demonstrate ZFNs enable highly efficient targeted addition of several large XIST transgenes (up to 21 kb) into multiple selected loci of human genome, which are the largest sequences for targeted addition to date. Surprisingly, the frequency of targeted integration is independent of the size of transgenes but possibly depends on ZFN cleavage activity. Highly efficient targeted addition of large XIST transgenes allows to directly compare silencing of XIST transgenes that contain different functional domains in a pool of many independent integrants. This study provides a powerful tool to test functional mini-XIST transgenes that can be packaged in a viral vector, and has particularly important applications in potential development of in vivo delivery of XIST transgenes into an organ of patients with trisomic chromosomal abnormalities.

Developing genetic tools to exploit Chaetomium thermophilum for biochemical analyses of eukaryotic macromolecular assemblies.

We describe a method to genetically manipulate Chaetomium thermophilum, a eukaryotic thermophile, along with various biochemical applications. The transformation method depends on a thermostable endogenous selection marker operating at high temperatures combined with chromosomal integration of target genes. Our technique allows exploiting eukaryotic thermophiles as source for purifying thermostable native macromolecular complexes with an emphasis on the nuclear pore complex, holding great potential for applications in basic science and biotechnology.

A method for specifically targeting two independent genomic integration sites for co-expression of genes in CHO cells

Stable mammalian production cell lines in suspension culture enable the reproducible expression of target genes in any desired scale using bioreactor technology. Targeted integration methods have been developed to cut down timelines for the generation of stable producer cell lines. The powerful Flp recombinase mediated cassette exchange (RMCE) technique allows fast integration of target genes in preselected and optimized high expression loci in so called master cell lines. Up to now, these cells only enable the expression from a single locus. Here, we describe the set-up required for the generation of multiple tagged master cell lines on the example of a binary RMCE expression system in the glycosylation mutant CHO Lec3.2.8.1 cell line. We show how this technology is used for the expression of proteins from multiple loci by generating a binary RMCE expression system. The tools and strategy for the construction of binary master cell lines with different combinations of expression level are described in detail. The binary production cell lines show independent expression of the individual exchange loci of the producer cell lines. The expression level for the model protein tdTomato is the cumulative expression for the chosen combination of the expression loci of the master cell line. This binary RMCE expression system can be further developed to a multi RMCE expression system for co-expression of protein complex subunits with predetermined expression ratio of each individual exchange locus.

Identification of bottlenecks in antibody expression using targeted gene integration

Antibody engineering allows proper design of improved properties in antibody products such as a high binding affinity, stable biophysical properties and low immunogenicity. Besides these obvious features improved by elegant design the primary amino acid sequence also has tremendous effects on the performance of the expression host itself influencing cell culture parameters including specific productivities and growth rates therefore having major impact on the finally obtained antibody expression titers [1]. By understanding the underlying mechanisms how certain primary sequence combinations influence culture performance in conjunction with biophysical features of the antibody protein, we might rationally improve the production process of these expensive but essential biotherapeutic products. Many different antibody variants were expressed in our group in different projects based on various isotypes. For several antibody variable regions we could observe a direct correlation of advantageous cell performance and a high degree of similarity to the closest human germline sequence of the respective mature antibody leading to higher specific productivities. However, the fundamental principles of these consistent observations and the correlation of cell behavior and biophysical product properties remain quite elusive. Therefore, in this project we aim to set the proper foundations to investigate the generalization of a concept to improve cell performance and antibody expression based on the primary sequence at a high germinality degree (Figure ​(Figure1).1). To have proper control of transgene integration locus, gene copy numbers and mRNA transcript level a suitable host for recombinase-mediated cassette exchange (RMCE) was developed to compare different antibody variants under isogenic conditions. Open in a separate window Figure 1 Observed correlation of specific productivity and sequence identity to the closest human germline sequence (germinality) (A). Antibody expression levels are influenced by several factors. Recombinase-mediated cassette exchange was applied to control for gene copy number and mRNA transcript level. Low expressing antibody variants (2F5, 4B3 and 2G12) were compared to its closest human germline sequence together with a therapeutic antibody (Ustekinumab) with low degree of germinality (B). Respective germline antibody variants were constructed by combination of germline V-, D- and J-segments.

Cas9-Guide RNA Directed Genome Editing in Soybean.

Recently discovered bacteria and archaea adaptive immune system consisting of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) endonuclease has been explored in targeted genome editing in different species. Streptococcus pyogenes Cas9-guide RNA (gRNA) was successfully applied to generate targeted mutagenesis, gene integration, and gene editing in soybean (Glycine max). Two genomic sites, DD20 and DD43 on chromosome 4, were mutagenized with frequencies of 59% and 76%, respectively. Sequencing randomly selected transgenic events confirmed that the genome modifications were specific to the Cas9-gRNA cleavage sites and consisted of small deletions or insertions. Targeted gene integrations through homology-directed recombination were detected by border-specific polymerase chain reaction analysis for both sites at callus stage, and one DD43 homology-directed recombination event was transmitted to T1 generation. T1 progenies of the integration event segregated according to Mendelian laws and clean homozygous T1 plants with the donor gene precisely inserted at the DD43 target site were obtained. The Cas9-gRNA system was also successfully applied to make a directed P178S mutation of acetolactate synthase1 gene through in planta gene editing.

Chloroplast localization of Cry1Ac and Cry2A protein--an alternative way of insect control in cotton.

BackgroundInsects have developed resistance against Bt-transgenic plants. A multi-barrier defense system to weaken their resistance development is now necessary. One such approach is to use fusion protein genes to increase resistance in plants by introducing more Bt genes in combination. The locating the target protein at the point of insect attack will be more effective. It will not mean that the non-green parts of the plants are free of toxic proteins, but it will inflict more damage on the insects because they are at maximum activity in the green parts of plants.ResultsSuccessful cloning was achieved by the amplification of Cry2A, Cry1Ac, and a transit peptide. The appropriate polymerase chain reaction amplification and digested products confirmed that Cry1Ac and Cry2A were successfully cloned in the correct orientation. The appearance of a blue color in sections of infiltrated leaves after 72 hours confirmed the successful expression of the construct in the plant expression system. The overall transformation efficiency was calculated to be 0.7%. The amplification of Cry1Ac-Cry2A and Tp2 showed the successful integration of target genes into the genome of cotton plants. A maximum of 0.673 μg/g tissue of Cry1Ac and 0.568 μg/g tissue of Cry2A was observed in transgenic plants. We obtained 100% mortality in the target insect after 72 hours of feeding the 2nd instar larvae with transgenic plants. The appearance of a yellow color in transgenic cross sections, while absent in the control, through phase contrast microscopy indicated chloroplast localization of the target protein.ConclusionLocating the target protein at the point of insect attack increases insect mortality when compared with that of other transgenic plants. The results of this study will also be of great value from a biosafety point of view.

Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway.

Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for production of therapeutic proteins. However, development of recombinant CHO cell lines has been hampered by unstable and variable transgene expression caused by random integration. Here we demonstrate efficient targeted gene integration into site-specific loci in CHO cells using CRISPR/Cas9 genome editing system and compatible donor plasmid harboring a gene of interest (GOI) and short homology arms. This strategy has enabled precise insertion of a 3.7-kb gene expression cassette at defined loci in CHO cells following a simple drug-selection, resulting in homogeneous transgene expression. Taken together, the results displayed here can help pave the way for the targeting of GOI to specific loci in CHO cells, increasing the likelihood of generating isogenic cell lines with consistent protein production.

An improved Tet-on system to tightly conditionally regulate reporter gene expression

Reporter genes are often used as markers to track the integration and expression of target genes in animal genetic engineering. To avoid potential side effects from reporter genes, in this study an improved Tet-on system was developed to control reporter gene expression, and its effectiveness was explored in transgenic cells. First, the rtTA protein was fused with Tat and NLS proteins to obtain the prokaryotic expression vector pET32a-Tat-rtTA-NSL. A eukaryotic transgenic vector was constructed, p-HS4-BPA-TmA-HS4, in which the reporter (mCherry) and target (PRL) genes were promoted by TRE and BCN, respectively. After confirming the functionality of the transgenic vector, purified rtTA protein and Dox were added to induce expression of the mCherry gene. The optimal amount of purified rtTA protein, its influence on target gene expression, and the time of rtTA protein action were each investigated separately. The results showed that rtTA protein was expressed in transformed E. coli with IPTG induction. TRE could promote mCherry gene expression by co-transfecting the constructed transgenic vector and prtTA plasmid. When purified rtTA protein and Dox were added, red fluorescence was observed in Bcap-37 cells transfected with the p-HS4-BPA-TmA-HS4 vector, and the exogenous PRL gene was expressed regardless of mCherry gene expression. The optimal amount of rtTA protein was 16 μg/mL, and it needed about 6 h to promote mCherry gene expression in transfected cells. These results demonstrate that the expression of the mCherry reporter gene can be tightly and conditionally regulated by our Tet-on system.

358 EXCESS EXPRESSION OF 11β-HSD1 CONTRIBUTES TO LETHALITY THROUGH DYSFUNCTION IN ENERGY BALANCE BETWEEN ANABOLIC PROCESS AND ENERGY RECOVERY PROCESS

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) effectively amplifies glucocorticoid action in liver, adipose tissue, and brain. This enzyme converts the inactive 11-keto form into a glucocorticoid (e.g. cortisol). Glucocorticoids (GC) are steroid hormones found in the body and produced by the adrenal cortex, the outer layer of the adrenal glands. Glucocorticoids are lipophilic and readily access their intracellular receptors. Glucocorticoids regulate carbohydrate, fat, and protein metabolism. In a previous study, we constructed a vector composed of 2 parts: the 11β-HSD1 expression cassette and the selection cassette containing EGFP and Neo resistant genes. Porcine fibroblasts overexpressing 11β-HSD1 under the control of adipose tissue-specific aP2 promoter were established and used in somatic cell nuclear transfer (SCNT). Somatic cells from the resulting stillborn transgenic piglets were used in a second round of SCNT. Non-obese, transgenic piglets overexpressing 11β-HSD1 were obtained and were identified through PCR-based methods using specific primers for the targeting cassettes from the genomic DNA of piglets. Six live piglets, 1 stillborn piglet, and 3 mummies were born. Integration of target gene into the genomic DNA was confirmed for all of them. However, all 6 live piglets died within 1 month. All of the piglets had displayed hypoglycemia. Increased expression of 11β-HSD1 in metabolic tissues induced up-regulation of gluconeogenesis-related genes (G6PT, G6Pase, PEPCK, HNF4a, FOXO1) in liver and kidney, and showed up-regulation of lipogenesis-related genes (SREBP1c, FASN, DGAT, ACC, SCD) in muscle. The AMPK and SIRT signalling, which controls energy balance and mitochondrial biogenesis, was also stimulated. We propose that overexpression of 11β-HSD1 evokes the excess production and action of glucocorticoid or its receptors, and activates gluconeogenic and lipogenic pathways. For this reason, AMPK and SIRT1 signalling was induced. Also, in compensation of energy loss by anabolic processes, the expression of mitochondrial biogenesis-related genes was increased. Finally, the constitutive expression of 11β-HSD1 might continuously activate complementary energy-gaining processes, and these problems could develop into more fatal diseases that resulted in the piglets' death.

28 EFFECTS OF RECLONING ON THE PRODUCTION OF PIGS OVEREXPRESSING 11 β-HYDROXYSTEROID DEHYDROGENASE TYPE 1 (11β-HSD1)

The incidence of metabolic syndrome is increasing globally, as the prevalence of obesity continues to rise. However, the basic mechanisms of metabolic syndrome are not completely known yet. Therefore, animal disease models are required for the study of metabolic syndrome. The overexpression of 11 β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in mice leads to metabolic syndrome; thus, we attempted to produce pigs with overexpression of 11β-HSD1 gene by somatic cell nuclear transfer (SCNT). However, low transgenic (TG) efficiency has been an obstacle to the production of TG pigs. A SCNT method in which somatic cells derived from TG pig are used as the nuclear donor (re-cloning method) is an effective technique for TG pig production. In this study, we attempted to increase TG efficiency by the re-cloning method. Pregnancy efficiency, production efficiency, and TG efficiency were compared with sources of donor cells (transfected TG fetal fibroblast v. TG fibroblast derived from newborn TG cloned pig). A total of 1382 and 881 TG SCNT embryos were produced from fetal fibroblast v. cloned fibroblast, and then transferred to 13 and 10 recipients. The pregnancy rate was not significantly different (30.8% v. 20.0%). Seventeen live piglets and 5 stillborn piglets were born from 4 recipients in the fetal fibroblast group, and 8 live piglets, 2 stillborn piglets, and 3 mummies were born from 2 recipients in the cloned fibroblast group. There were no significant differences in the production efficiency (3.7% v. 5.0%). All of the 13 re-cloned piglets showed reporter and target gene integration. But, of 22 fetal fibroblast-cloned piglets, reporter gene integration was confirmed in 9, but only 3 clone piglets showed reporter gene integration. Efficiency of TG was significantly increased in re-cloning group (13.6% v. 100.0%). In this study, TG efficiency of 11β-HSD1 overexpressed pigs was improved by re-cloning method. These results indicate that re-cloning is an efficient method for production of TG cloned pigs.This work was supported by a grant from the Next-Generation Bio Green 21 Program (No. PJ009563032014), Rural Development Administration, Republic of Korea.

Dengue virus E glycoprotein production in transgenic rice callus.

Dengue is a disease caused by dengue virus and represents the most important arthropod-borne viral disease in humans. Dengue virus enters host cells via binding of envelope glycoprotein (E) to a receptor. In this study, plant expression vectors containing native and synthetic glycoprotein E genes (sE) modified based on plant-optimized codon usage and fused with an ER retention signal were constructed under control of the rice amylase 3D promoter expression system. Plant expression vectors were introduced into rice callus (Oryza sativa L. cv. Dongin) via particle bombardment-mediated transformation. The integration and expression of target genes were confirmed in the transgenic callus by genomic DNA PCR and Northern blot analyses, respectively. The plant-codon optimized sE gene with an ER retention signal showed high protein production levels based on Western blot analysis of approximately 18.5ug/g dried calli weight by immunoblot-based densitometric analysis. These results suggest that the plant-codon optimized sE gene with an ER retention signal was highly produced in the transgenic rice callus.