408 FOREIGN GENE INTEGRATION PATTERNS IN TRANSGENIC PORCINE FETUSES PRODUCED BY ICSI-MEDIATED GENE TRANSFER

The expression of foreign gene in transgenic animals produced by pronuclear microinjection is often confounded by the position effects caused by not only the nature of chromosomal integration site but also the number and arrangement of multiple transgene copies. Gene targeting provides a new way to overcome these inhibitions by introducing single-copy transgene into a chosen site. The choice of a good chromosomal site will favor transgene expression in a predictable fashion. In this study, we tested a new site (fgfr-4) for foreign gene integration and expression. A t-PA mutant (t-PAm) expression cassette under bovine alphas1-casein regulatory sequences was efficiently knocked-in fgfr-4 site through homologous recombination. The t-PAm was expressed in the milk of all targeted mice. Our experiment indicates that the fgfr-4 may be a candidate site for knocking foreign gene to make transgenic animals.

Intracytoplasmic sperm injection (ICSI) of DNA-binding sperm produces transgenic offspring as effectively as pronuclear microinjection (PNM). A significant difference in these two methods is that DNA is introduced into MII oocytes during ICSI, which is likely to allow earlier gene integration compared to PNM. This leads us to hypothesize that ICSI reduces the chance of development of a mosaic embryo, a mixture of transgene-positive and -negative cells. To test this hypothesis, we compared expression patterns of the green flourescent protein (GFP) gene introduced by ICSI and PNM into murine and porcine oocytes. For ICSI, 2 to 5 × 105/μL of sperm frozen-thawed in CZB (for mice) or NIM (for pigs) were co-incubated with 2.5 ng/μL of transgene fragments (CAG-EGFP; 3 kb) for 5 min. Murine sperm were microinjected into in vivo-matured oocytes, and porcine sperm into in vitro-matured oocytes. PNM was performed by microinjection of several picoliters of the transgene fragments (10 ng/μL) into pronuclei of in vivo-fertilized oocytes for mice and in vitro-matured and -fertilized oocytes for pigs. ICSI and PNM embryos were cultured in vitro to the morula stage and treated with 0.5% pronase to remove the zona pellucida. These morulae were disassembled into individual blastomeres by pipetting into PBS containing 100 μM EDTA and examined for GFP expression under fluorescence microscopy. As shown in Table 1, the rate of mosaicism in GFP-expressing embryos was significantly lower for ICSI than for PNM (P < 0.01). In addition, GFP-expressing ICSI embryos were likely to contain high percentages, 81 to 100%, of GFP-positive cells, whereas GFP-expressing PNM embryos were significantly less likely to contain such high percentages of GFP-positive cells (P < 0.01). From these results, we conclude that transgenesis by ICSI was less likely to produce mosaic embryos, and that produced transgenic embryos contained higher proportions of transgene-positive cells, although genomic integration remains to be determined. Table 1. Transgene expression by ICSI and pronuclear microinjection in murine and porcine embryos This work was supported by PROBRAIN.

Additional virulence genes influence transgene expression: transgene copy number, integration pattern and expression

Manipulating piggyBac Transposon Chromosomal Integration Site Selection in Human Cells

Transgene expression in stably transgenic organisms is affected by many factors, including the copy number of the transgene in the genome and by interactions between the transgene and flanking DNA sequences. Very high transgene copy number has also been shown to affect genetic stability in transgenic plants and animals. Two commonly used methods for transfecting cells prior to their use in nuclear transfer (NT) are liposome-mediated transfection and electroporation. Little is known about the transgene copy number or variability of the copy number with these techniques. The objective of this study was to determine transgene copy number after liposome-mediated transfection and electroporation. The mean transgene copy number and variability between individual integration events have been determined. Q-PCR conditions were optimized for primer annealing temperature and concentration when amplifying a region of a plasmid expressing green fluorescent protein (GFP) under the control of the human elongation factor (hEF) promoter (hEFGFP) used for transfection. The quantitative nature of the Q-PCR reaction was confirmed by amplifying 10-fold dilutions of the plasmid and plotting the threshold cycle (CT) value against the log of the plasmid concentration. A correlation coefficient of 1.00 and a calculated PCR efficiency of 93.3% were obtained from this analysis. Caprine fibroblasts were transfected by electroporation with 20 μg of DNA or FuGENE® HD (Roche, Nutley, NJ, USA) reagent with 6 μg of DNA using either a circular or linearized hEFGFP plasmid. Transformed cells were plated at low density in medium containing Geneticin® (Gibco, Grand Island, NY USA). After 10 days of culture, single-cell colonies were isolated and expanded. When cultures reached 1 to 2 million cells, genomic DNA was isolated. Transgene copy number was determined by amplifying genomic DNA from individual clones representing 1 × 105 cells with Q-PCR. Transgene copy number was calculated from a standard curve of the transgene plasmid. The mean transgene copy number for electroporation circular was 2.7 ± 0.75 (n = 32 colonies) and 1.3 ± 0.65 (n = 19) when using a linear DNA construct. FuGENE HD using a circular plasmid construct generated a mean gene copy number of 0.5 ± 0.11 (n = 14) and 0.64 ± 0.13 (n = 16) for the linear plasmid construct. One-way ANOVA followed by multiple pair-wise comparisons using Tukey’s method showed significant differences when comparing electroporation circular to all other treatments. However, there were no differences when comparing electroporation linear, FuGENE HD circular, and FuGENE HD linear to each other. Because the calculated mean copy number for transfection with FuGENE HD was consistently less than 1, it is assumed that these colonies consisted predominantly of single-copy integrations. Our results indicate that the transfection method can affect gene copy number. Electroporation resulted in multiple but few copies whereas Fugene HD resulted in predominantly single-copy integrations. The probability of transgene mutation with single-copy integration suggests that electroporation is preferable forproducing transgenic animals by NT.

Confirmation of Drought Tolerance of Ectopically Expressed AtABF3 Gene in Soybean.

The strength of recombinant gene expression is a key property of cell lines for biopharmaceutical protein production. In most stable cell lines the expression vector is stably introduced into the host chromosomal DNA. Apart from the copy number and the used expression control elements the performance of recombinant expression vectors is modulated by genetic and epigenetic features provided by flanking host elements. Since targeted integration is very difficult cell clones with high expression of a recombinant vector are created by random integration and large scale screening for gene expression. This allows the isolation of those rare recombinant cells in which gene expression is optimal. This is usually due to locus-specific influences of the chromosomal surroundings. We have developed an efficient methodology for targeting expression cassettes to specific chromosomal sites [1,2]]. The method (Flp recombinase mediated cassette exchange - RMCE) allows the repeated use of defined loci by targeting constructs for expression of proteins and viruses, thereby allowing to exploit the positive features of a given integration site [3,4]]. Thereby, a systematic evaluation of the performance of a set of expression vectors in various chromosomal sites becomes feasible. In this study we screened for high performance integration sites in HEK293 and CHO-K1 cells supporting expression cassettes driven by a potent promoter. As a read out, production of antibodies and recombinant retroviral vectors were used. Thereby, we could show that high level expression of a given promoter is restricted to defined integration sites, while other sites show only moderate expression (data not shown). An important new finding was that a given chromosomal site is not flexible with respect to the integrated cassette but requires the integration of specific promoters. As illustrated in figure ​figure1,1, an integration site, identified for supporting high level expression of an SV40 promoter driven cassette fails to adequately support expression of MPSV and adenoviral major late promoter (AdmlP). Vice versa, another site, initially screened for supporting MSCV promoter expression, could restore expression of the highly homologous MPSV promoter while the SV40 promoter and the AdmlP promoter give only moderate expression. Figure 1 Mode of tagging defines the optimal targeting cassette. Two highly potent chromosomal integration sites were screened upon random integration of an expression cassette driven by the SV40 promoter and the MSCV promoter, respectively. By means of Flp recombinase ... Finally, we tested the impact of the orientation of the cassette in a specific chromosomal site. We found that some integration sites are flexible with respect to the orientation of the expression cassettes while others support expression only in one direction (data not shown). While classical enhancer elements are known to activate promoters largely independent from the relative position this finding suggests that other cis acting elements affect the incoming cassettes in an orientation dependent manner. Together, this shows that not the nature of integration site and the design of the vector as such define the performance of a producer cell clone. Rather, the interplay between these components defines the level and stability of expression. Since these interactions cannot be predicted, the performance of a vector in a given site has to be evaluated empirically. In order to exploit favourable sets of chromosomal sites and vectors we made use of bacterial artificial chromosome (BAC) vectors. By recombineering, expression cassettes were integrated into pre-selected chromosomal sites as encoded by BAC vectors. These vectors were randomly integrated into cells by standard transfection protocols. Clones were isolated and evaluated for expression. As expected, highly reproducible expression characteristics were found in individual clones (data not shown). In conclusion, the definition of favorable combinations of specific integration sites and vector design allow the rational exploitation of given chromosomal sites. For this purpose, technologies for site specific genetic manipulation of mammalian cells are essential. This concerns both targeted integration of expression cassettes into defined loci (such as RMCE or site specific nuclease induced homologous recombination) or by transduction of large chromosomal domains (as provided by BAC vectors). These technologies pave the way for predictable and high expression of biotechnologically relevant products such as antibodies and recombinant viral vectors.

BackgroundSynechocystis sp. PCC 6803 provides a well-established reference point to cyanobacterial metabolic engineering as part of basic photosynthesis research, as well as in the development of next-generation biotechnological production systems. This study focused on expanding the current knowledge on genomic integration of expression constructs in Synechocystis, targeting a range of novel sites in the chromosome and in the native plasmids, together with established loci used in literature. The key objective was to obtain quantitative information on site-specific expression in reference to replicon copy numbers, which has been speculated but never compared side by side in this host.ResultsAn optimized sYFP2 expression cassette was successfully integrated in two novel sites in Synechocystis chromosome (slr0944; sll0058) and in all four endogenous megaplasmids (pSYSM/slr5037-slr5038; pSYSX/slr6037; pSYSA/slr7023; pSYSG/slr8030) that have not been previously evaluated for the purpose. Fluorescent analysis of the segregated strains revealed that the expression levels between the megaplasmids and chromosomal constructs were very similar, and reinforced the view that highest expression in Synechocystis can be obtained using RSF1010-derived replicative vectors or the native small plasmid pCA2.4 evaluated in comparison. Parallel replicon copy number analysis by RT-qPCR showed that the expression from the alternative loci is largely determined by the gene dosage in Synechocystis, thereby confirming the dependence formerly proposed based on literature.ConclusionsThis study brings together nine different integrative loci in the genome of Synechocystis to demonstrate quantitative differences between target sites in the chromosome, the native plasmids, and a RSF1010-based replicative expression vector. To date, this is the most comprehensive comparison of alternative integrative sites in Synechocystis, and provides the first direct reference between expression efficiency and replicon gene dosage in the context. In the light of existing literature, the findings support the view that the small native plasmids can be notably more difficult to target than the chromosome or the megaplasmids, and that the RSF1010-derived vectors may be surprisingly well maintained under non-selective culture conditions in this cyanobacterial host. Altogether, the work broadens our views on genomic integration and the rational use of different integrative loci versus replicative plasmids, when aiming at expressing heterologous genes in Synechocystis.

Biosafety Assessment of Site-directed Transgene Integration in Human Umbilical Cord–lining Cells

BackgroundAs compared with traditional transgene copy number detection technologies such as Southern blot analysis, real-time PCR provides a fast, inexpensive and high-throughput alternative. However, the real-time PCR based transgene copy number estimation tends to be ambiguous and subjective stemming from the lack of proper statistical analysis and data quality control to render a reliable estimation of copy number with a prediction value. Despite the recent progresses in statistical analysis of real-time PCR, few publications have integrated these advancements in real-time PCR based transgene copy number determination.ResultsThree experimental designs and four data quality control integrated statistical models are presented. For the first method, external calibration curves are established for the transgene based on serially-diluted templates. The Ct number from a control transgenic event and putative transgenic event are compared to derive the transgene copy number or zygosity estimation. Simple linear regression and two group T-test procedures were combined to model the data from this design. For the second experimental design, standard curves were generated for both an internal reference gene and the transgene, and the copy number of transgene was compared with that of internal reference gene. Multiple regression models and ANOVA models can be employed to analyze the data and perform quality control for this approach. In the third experimental design, transgene copy number is compared with reference gene without a standard curve, but rather, is based directly on fluorescence data. Two different multiple regression models were proposed to analyze the data based on two different approaches of amplification efficiency integration. Our results highlight the importance of proper statistical treatment and quality control integration in real-time PCR-based transgene copy number determination.ConclusionThese statistical methods allow the real-time PCR-based transgene copy number estimation to be more reliable and precise with a proper statistical estimation. Proper confidence intervals are necessary for unambiguous prediction of trangene copy number. The four different statistical methods are compared for their advantages and disadvantages. Moreover, the statistical methods can also be applied for other real-time PCR-based quantification assays including transfection efficiency analysis and pathogen quantification.

Through cold acclimation, plant can increase its tolerance capability upon exposure to low temperature. Previous study showed that CBFs/DREBs (C-repeat binding factor/ dehydration response element binding factor) were the major transcription factors that involved in cold acclimation process. The function of CBFs/DREBs evolved highly conserve between dicot and monocot. Recent study from Arabidopsis has defined ICE1 (inducer of CBF expression), a bHLH (basic helix-loop-helix) protein, as an important transcriptional factor that acts on the promoter of CBF gene and regulates its expression. In this study, based on rice functional genomic approach with transgenic rice analysis, we aimed to understand physiological function of Arabidopsis AtICE1 and Barly HvICE1 under different abiotic stresses. Meanwhile, the action mold of AtICE1 and HvICE1 will be compared under various stresses. To reach this goal, first, by bioinformatics search at least four of OsICE genes were found in rice genome. From currently available rice microarray data revealed that OsICE1 expression was highly induced by salt and drought but not affected in low temperature. OsICE2 and OsICE3 transcripts were repressed upon exposure to salt and drought environment. On the other hand, OsICE4 expression was salt and drought induced. Besides, OsICE3 and OsICE4 gene expression were both increased under low temperature stress. Then, we used TNG67 (Oryza sativa L., japonica; cold and salt tolerant but drought sensitive) and TCN1 (Oryza sativa L., indica; cold and salt sensitive but drought resistant) rice cultivars to investigate OsICEs; OsDREBs and OsDREBs regulon-related downstream genes expression profiles under abiotic stress treatments. The results indicated that OsICE2 expression level were down-regulated quickly in TCN1 at low temperature. And the amount of OsDREB1F、OsDREB1G、OsDREB1H、OsDREB1I and OsDREB1J expressions in TCN1 were also less than those of TNG67. To further elucidate the physiological effects of AtICE1 and HvICE1 under various abiotic stresses, we generated ICEs-overexpressed transgenic rice lines, 35S::AtICE1 and 35S::HvICE1. By Southern blotting analysis, TAIL-PCR, and PCR-based genotyping, we determined the copy numbers of transgene, T-DNA inserted flanking sequence and obtained either one or two copies of homozygous transgenic lines. RT-PCR result showed under normal growth condition indeed we can detect the overexpression of ICE genes in 35S::AtICE1 and 35S:: HvICE1 transgenic rices. However; compared to low-temperature stress-treated wild type plant, the whole gene expression profile of ICE-corresponding downstream genes (OsDREBs) did not obviously changed. The physiological analysis of abiotic stress tolerance assay, including chlorophyll, malondialdehyde (MDA) and proline content measurements showed that 35S::AtICE1 transgene rice with OsDREB1A; 1B; 1C and 2B transcripts enhanced could increase cold tolerance but not for drought and salt tolerance. 35S::HvICE1 transgene rice that with slightly OsDREB 1B; 1C and 1E gene expression increased could raise up its drought and cold tolerance but not salt tolerance. Taken together, the above results suggested that AtICE1 and HvICE1 may function not exactly the same in cold acclimation pathway. This may due to other ICEs co-operate in the regulation of CBFs/DREBs and CBFs/DREBs regulon-related gene expression or ICEs activity can be adjusted through post-translation modifications that lead to different responses when exposure to different abiotic stresses.

Spontaneous germline amplification and translocation of a transgene array

Transgenic (Tg) pigs expressing a fluorescent protein are extremely useful for research into transplantation and regenerative medicine. This study aimed to create Tg pigs expressing monomeric Plum (mPlum), a far-red fluorescent protein with a longer wavelength than enhanced green fluorescent protein (EGFP) and humanized Kusabira Orange (huKO), the two fluorescent proteins that have been used previously for Tg pig production. A linearized CAG-mPlum transgene construct was transferred into porcine fetal fibroblasts (PFF) by electroporation. mPlum fluorescence-positive cells were collected using a cell sorter and used as nuclear donors (mPlum-PFF) for somatic cell nuclear transfer (SCNT). In vitro-matured oocytes were obtained from porcine cumulus–oocyte complexes cultured in NCSU23-based medium and were used to obtain recipient oocytes for SCNT after enucleation. Then, SCNT was performed as reported previously (Matsunari et al., 2008). The reconstructed embryos were cultured for 7 days in porcine zygote medium-5 (PZM-5). mPlum fluorescence expression was screened during the early development of the embryos. After 5 or 6 days of culture, the SCNT embryos were surgically transferred to the uterus of a recipient gilt. We first obtained fetuses on Day 36 or 37 of gestation by Caesarean section and the PFF were retrieved from their skin. Fluorescence expression was analysed using fluorescence microscope, and the number of transgene copies in each fetus was determined by Southern blot analysis. We also analysed whether unique spectral properties of mPlum are suitable for multicolor imaging using confocal microscope and flow cytometer. The identification of mPlum-expressing PFF under the mixed culture of PFF expressing EGFP and huKO was examined. The 2 cell lines of PFF expressing EGFP and huKO were previously generated in our laboratory. Rates of normal cleavage and blastocyst formation occurred in the SCNT embryos generated with mPlum-PFF (mPlum embryos) were equivalent to those of SCNT embryos derived from nontransgenic PFF (34/42, 81.0%; 33/42, 78.6% v. 37/40, 92.5%; 30/40, 75.0%). Total cell numbers in mPlum and control blastocysts did not differ significantly (88.3 ± 6.0 v. 99.9 ± 8.8). Fluorescence expression in the mPlum embryos began at the 8-cell stage and became brighter from the morula stage. The gilt into which 103 mPlum embryos were transferred produced 3 fetuses. These fetuses expressed mPlum fluorescence systemically and had 1 to 5 copies of the transgene. Multicolor fluorescence imaging and flow cytometric analyses of a mixed culture of mPlum PFF and PFF expressing EGFP and huKO showed that clear identification and isolation of cells displaying each of the 3 fluorescence signals was possible. These observations demonstrate that the transfer of CAG-mPlum did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of Tg cloned pigs that systemically expressed mPlum. This work was supported by JSPS KAKENHI Grant Number 25293279.

It has been demonstrated that transgene expression is associated with copy number in transgenic animals. Here in, we generated 7 genetically modified pigs expressing both soluble human tumour necrosis factor receptor type Ig-Fc (shTNFRI-Fc) and human heme oxygenase-1 (HO-1). 1 day after Caesarean section, all transgenic cloned piglets showed postnatal death. In the present study, the transgene copy number, H2O2 and superoxide dismutase (SOD) levels in cloned piglet liver were examined to identify the relationship between transgene copy number and oxidative stress of postnatal liver. In this study, 2,209 cloned embryos using somatic cells with 15 copies of shTNFRI-Fc and HO-1 were produced by somatic cell nuclear transfer, and transferred into 6 synchronized recipient sows. Among them, pregnancies were identified in 4 recipients using ultrasonography and only 1 recipient was maintained until full term. In total, 7 cloned piglets were delivered by the Caesarean section. On the next day, they showed postnatal death with clinical symptoms such as dyspnea (Group A). As control group, 292 cloned embryos produced from the cells with at least 4 copies of 2 transgenes shTNFRI-Fc and HO-1 were transferred into a synchronized recipient and pregnancy was identified. Two cloned piglets were delivered normally and maintained healthy. The liver of a live cloned piglet with at least 4 copies (Group B) at 2 days after the Caesarean section was isolated and compared with those of dead 7 cloned piglets (Group A) for HO-1, shTNFRI-Fc, H2O2, and SOD by ELISA analysis. The transgene copy number and expression of shTNFRI-Fc and HO-1 were confirmed by genomic DNA PCR, quantitative real-time PCR (qRT-PCR) and ELISA with appropriate antibodies. Statistical analysis was performed using Graphpad Prism. Level of HO-1, shTNFRI-Fc, H2O2, and SOD ELISA results of each piglets were analysed by unpaired t-test with Welch’s correction. While a transgenic piglet (Group B) had at least 4 copy numbers, all dead cloned piglets (Group A) showed 15 copy numbers. A high level of transgene HO-1 and shTNFRI-Fc expression of liver-derived cells in cloned piglets (Group A) was significantly identified compared with those of a transgenic piglet (Group B) by qRT-PCR and ELISA. While the H2O2 level in cloned piglet liver with 15 copy numbers (Group A) was significantly higher (P < 0.05), the SOD level was lower than those of a cloned pig (Group B; P < 0.05). These results demonstrated that multiple copy numbers could affect the level of oxidative stress in cloned piglet liver. It also affected the transgene expression levels and mortality of cloned piglets. This study was supported by National Research Foundation (#2015R1C1A2A01054373. 2016M3A9B6903410), Ministry of Trade, Industry & Energy (#10048948), Korea Institute of Planning and Evaluation for Technology in food, agriculture, forestry and fisheries (#114059–03–2-SB010), Research Institute for Veterinary Science, Natural Balance and the BK21 plus program.

Presence of multiple copies of a transgene has been found to associate with gene silencing that may manifest early or only over a period of time. As such, transgenic copy number should be analyzed as soon as possible. The commonly used method to analyze gene copy number, Southern blotting, has been found to be unreliable for determination of gene copy number when rearrangement or tandem repeats integration occurred. In recent years, a powerful real-time fluorescence quantitative real-time PCR (qRT-PCR) method has been used to analyze gene copy number in genetically modified plants, but these methods have also their own application conditions. Thus, based on real-time quantitative PCR technology, the modified 2-△△CT method, which has been used usually in analyzing gene expression, was used for the first time, to analyze transgenic copy numbers in transgenic cotton. In this paper, a single-copy homozygous transgenic plant whose identity was confirmed by Southern blotting using restriction enzymes was used as a calibrator. Genomic DNA that was extracted from the calibrator and six transgenic T0 cotton plants transformed by pollen tube pathway or gene gun bombardment was amplified by quantitative PCR, and the gene copy numbers were estimated by the modified 2-△△CT method to be 6, 3, 2, 2, 3 and 2 copies. In addition, except 6 copies all other identifications (3, 2, 2, 3 and 2) were equally indicated by the corresponding Southern blotting. This study supports the modified 2-△△CT method using quantitative real-time PCR technology to provide a fast, high-throughput method to analyze the copy number of foreign genes in a large transgenic T0 population. Key words: Cotton, transgene copy number, modified 2-△△CT method, Southern blotting.