Concatemer-Associated Transgene Expression Patterns in Transgenic Marine Medaka Oryzias dancena Strains

Cho, Young Sun,Kim, Dong Soo,Nam, Yoon Kwon The Korean Society of Fisheries and Aquatic Scienc 2015 Fisheries and Aquatic Sciences Vol.18 No.1

To examine the interrelationship between transgenic insertion patterns and transgene expression profiles in established transgenic fish lines, four stable transgenic marine medaka Oryzias dancena germlines harboring ${\beta}$-actin regulator-driven RFP reporter constructs were selected. The established transgenic strains were characterized with regard to their transgenic genotypes (insertion pattern, concatemer formation, and transgene copy number based on genomic Southern blot hybridization and qPCR assay) and expression characteristics at the mRNA (qRT-PCR), protein (western blot), and phenotypic (fluorescent appearance) levels. From comparative examinations, it was found that transgenic expression at both the transcription and translation levels could be significantly downregulated in transgenic strains, potentially through methylation-mediated transgene silencing that was particularly associated with the formation of a long tail-to-head tandem concatemer in the chromosomal integration site(s). When this occurred, an inverse relationship between the transgene copy number and fluorescence intensity was observed in the resultant transgenic fish. However, with the other transgenic genotype, transgenic individuals with an identical Southern blot hybridization pattern, containing a tandem concatemer(s), had very different expression levels (highly robust vs. low expression strengths), which was possibly related to the differential epigenetic modifications and/or degrees of methylation. The concatemer-dependent downregulation of transgene activity could be induced in transgenic fish, but the overall pattern was strain-specific. Our data suggest that neither a low (or single) transgene copy number nor tandem transgene concatemerization is indicative of strong or silenced transgene expression in transgenic fish carrying a ubiquitous transgene. Hence, a sufficient number of transgenic lineages, with different genotypes, should be considered to ensure the establishment of the best-performance transgenic line(s) for practical applications.

Concatemer-Associated Transgene Expression Patterns in Transgenic Marine Medaka Oryzias dancena Strains

조영선,김동수,남윤권 한국수산과학회 2015 Fisheries and Aquatic Sciences Vol.18 No.1

To examine the interrelationship between transgenic insertion patterns and transgene expression profiles in established transgenic fish lines, four stable transgenic marine medaka Oryzias dancena germlines harboring β-actin regulator-driven RFP reporter constructs were selected. The established transgenic strains were characterized with regard to their transgenic genotypes (insertion pattern, concatemer formation, and transgene copy number based on genomic Southern blot hybridization and qPCR assay) and expression characteristics at the mRNA (qRT-PCR), protein (western blot), and phenotypic (fluorescent appearance) levels. From comparative examinations, it was found that transgenic expression at both the transcription and translation levels could be significantly downregulated in transgenic strains, potentially through methylation-mediated transgene silencing that was particularly associated with the formation of a long tail-to-head tandem concatemer in the chromosomal integration site(s). When this occurred, an inverse relationship between the transgene copy number and fluorescence intensity was observed in the resultant transgenic fish. However, with the other transgenic genotype, transgenic individuals with an identical Southern blot hybridization pattern, containing a tandem concatemer(s), had very different expression levels (highly robust vs. low expression strengths), which was possibly related to the differential epigenetic modifications and/or degrees of methylation. The concatemer-dependent downregulation of transgene activity could be induced in transgenic fish, but the overall pattern was strain-specific. Our data suggest that neither a low (or single) transgene copy number nor tandem transgene concatemerization is indicative of strong or silenced transgene expression in transgenic fish carrying a ubiquitous transgene. Hence, a sufficient number of transgenic lineages, with different genotypes, should be considered to ensure the establishment of the best-performance transgenic line(s) for practical applications.

Original Article : Concatemer-Associated Transgene Expression Patterns in Transgenic Marine Medaka Oryzias dancena Strains

( Young Sun Cho ),( Dong Soo Kim ),( Yoon Kwon Nam ) 한국수산과학회(구 한국수산학회) 2015 Fisheries and Aquatic Sciences Vol.18 No.1

To examine the interrelationship between transgenic insertion patterns and transgene expression profiles in established transgenic fish lines, four stable transgenic marine medaka Oryzias dancena germlines harboring β-actin regulator-driven RFP reporter constructs were selected. The established transgenic strains were characterized with regard to their transgenic genotypes (insertion pattern, concatemer formation, and transgene copy number based on genomic Southern blot hybridization and qPCR assay) and expression characteristics at the mRNA (qRT-PCR), protein (western blot), and phenotypic (fluorescent appearance) levels. From comparative examinations, it was found that transgenic expression at both the transcription and translation levels could be significantly downregulated in transgenic strains, potentially through methylation-mediated transgene silencing that was particularly associated with the formation of a long tail-to-head tandem concatemer in the chromosomal integration site(s). When this occurred, an inverse relationship between the transgene copy number and fluorescence intensity was observed in the resultant transgenic fish. However, with the other transgenic genotype, transgenic individuals with an identical Southern blot hybridization pattern, containing a tandem concatemer(s), had very different expression levels (highly robust vs. low expression strengths), which was possibly related to the differential epigenetic modifications and/or degrees of methylation. The concatemer-dependent downregulation of transgene activity could be induced in transgenic fish, but the overall pattern was strain-specific. Our data suggest that neither a low (or single) transgene copy number nor tandem transgene concatemerization is indicative of strong or silenced transgene expression in transgenic fish carrying a ubiquitous transgene. Hence, a sufficient number of transgenic lineages, with different genotypes, should be considered to ensure the establishment of the best-performance transgenic line(s) for practical applications.

Production of Transgenic Pigs with an Introduced Missense Mutation of the Bone Morphogenetic Protein Receptor Type IB Gene Related to Prolificacy

Zhao, Xueyan,Yang, Qiang,Zhao, Kewei,Jiang, Chao,Ren, Dongren,Xu, Pan,He, Xiaofang,Liao, Rongrong,Jiang, Kai,Ma, Junwu,Xiao, Shijun,Ren, Jun,Xing, Yuyun Asian Australasian Association of Animal Productio 2016 Animal Bioscience Vol.29 No.7

In the last few decades, transgenic animal technology has witnessed an increasingly wide application in animal breeding. Reproductive traits are economically important to the pig industry. It has been shown that the bone morphogenetic protein receptor type IB (BMPR1B) A746G polymorphism is responsible for the fertility in sheep. However, this causal mutation exits exclusively in sheep and goat. In this study, we attempted to create transgenic pigs by introducing this mutation with the aim to improve reproductive traits in pigs. We successfully constructed a vector containing porcine BMPR1B coding sequence (CDS) with the mutant G allele of A746G mutation. In total, we obtained 24 cloned male piglets using handmade cloning (HMC) technique, and 12 individuals survived till maturation. A set of polymerase chain reactions indicated that 11 of 12 matured boars were transgene-positive individuals, and that the transgenic vector was most likely disrupted during cloning. Of 11 positive pigs, one (No. 11) lost a part of the terminator region but had the intact promoter and the CDS regions. cDNA sequencing showed that the introduced allele (746G) was expressed in multiple tissues of transgene-positive offspring of No.11. Western blot analysis revealed that BMPR1B protein expression in multiple tissues of transgene-positive $F_1$ piglets was 0.5 to 2-fold higher than that in the transgene-negative siblings. The No. 11 boar showed normal litter size performance as normal pigs from the same breed. Transgene-positive $F_1$ boars produced by No. 11 had higher semen volume, sperm concentration and total sperm per ejaculate than the negative siblings, although the differences did not reached statistical significance. Transgene-positive $F_1$ sows had similar litter size performance to the negative siblings, and more data are needed to adequately assess the litter size performance. In conclusion, we obtained 24 cloned transgenic pigs with the modified porcine BMPR1B CDS using HMC. cDNA sequencing and western blot indicated that the exogenous BMPR1B CDS was successfully expressed in host pigs. The transgenic pigs showed normal litter size performance. However, no significant differences in litter size were found between transgene-positive and negative sows. Our study provides new insight into producing cloned transgenic livestock related to reproductive traits.

Pollen-mediated flow of bar gene in transgenic herbicide-resistant turf grass Zoysia japonica

강홍규,정옥철,배태웅,선현진,송인자,박기웅,임평옥,이재천,이용억,송필순,이효연 한국식물생명공학회 2021 Plant biotechnology reports Vol.15 No.2

Weed control can be most efectively achieved through the use of herbicide-resistance transgene. A preliminary study bar�transgenic Zoysia japonica posed no serious risk on the unintended escape of the transgene from its cultivation site. The present follow-up investigation of the dispersion of pollen and its short-distance escape outside of the bar-transgenic Zoysia japonica habitats were ascertained in terms of environmental factors afecting anthesis and pollen viability. In a 24-h day cycle, zoysiagrass pollen was released predominantly between 08:00 and 10:00, and the pollen was most viable during the same time interval. Optimal temperature and humidity for pollen viability was 15–20 °C and 80–90%, respectively. The pollen germinated in 120 min after anthesis, but under cloudy conditions germination time doubled. No diferences in pol�len viability/longevity between the transgenic and non-transgenic plants were observed. The pollen-mediated gene fow of transgenic Zoysia japonica to wild-type non-transgenic zoysiagrass species was monitored by measuring the cross-over rate of the bar gene in the context of three diferent models. At distances within 5 m, the rate of gene fow ranged from 3 to 5.7% according to the models used. The greater the distance from the transgenic plant site, the lower the gene fow rate. The furthest transgene detected was 38 m away and exhibited a 0.25% gene fow rate. The radial model yielded a 3.7% escape rate within a 3 m radius and was wind direction dependent. The distance- and direction-dependent gene fow events were infuenced by wind direction and velocity during fowering season.

Triprimer-PCR method : Rapid and Reliable Detection of Transgenes in Transgenic rice plants

Lee, Dong-Keun,Seok, Soon Jong,Jang, In-Cheol,Hanm, Baek Hie,Kim, Ju-Jon Korean Society of Molecular Biology 1998 Molecules and cells Vol.8 No.1

Production of Transgenic Pigs Harboring the Human Erythropoietin (hEPO) Gene Using Somatic Cell Nuclear Transfer

CHO, Seong-Keun,HWANG, Kyu-Chan,CHOI, Yun-Jung,BUI, Hong-Thuy,NGUYEN, Van Thuan,PARK, ChangKyu,KIM, Jae-Hwan,KIM, Jin-Hoi Society for Reproduction and Development 2009 Journal of Reproduction and Development Vol.55 No.2

<P>The production of transgenic pigs using somatic cell nuclear transfer (scNT) has been widely described, but a technique for removing nontransfected donor cells and for creating different founder animals has not yet been fully elucidated. In this study, four different expression vectors (pBC1hEPO, pMARBC1hEPO, pBC1hEPOwpre and pMARBC1hEPOwpre) were compared to determine the highest transgene expression, ideal conditions of enrichment of recombinant cells <I>in vitro</I> and efficiency of transgenesis following transfection into HC11 mammary epithelial cells. The highest protein expression in HC11 cells was obtained from the pMARBC1hEPOwpre expression vector. Next, we evaluated the efficiency of transgenic pig production by using geneticin (G418) selection alone or by using real-time PCR selection following G418 selection. Ideal enrichment of recombinant cells was obtained by a combination of real-time PCR and G418 selection; embryos reconstructed using donor cells selected by a combination of real-time PCR and G418 selection gave rise to nine piglets, all of which were transgenic. Among them, three founder transgenic pigs were established. Exogenous DNA fragments were shown to be integrated into chromosomes 1q2.4, 1p2.3 and 6q2.4, respectively, in these three pigs. However, the transgenic rate using G418 selection alone was only 33% (two of six pigs) and showed a very low efficacy compared with that of the combination of real-time PCR and G418 selection. Our results provide a valuable experimental model for applying and evaluating transgenic technology in pigs.</P>

Effects of transgenic watermelon with CGMMV resistance on the diversity of soil microbial communities using PLFA

Yi, Hoon-Bok,Kim, Chang-Gi The Korean Society for Integrative Biology 2010 Animal cells and systems Vol.14 No.3

We compared the composition of phospholipid fatty acids (PLFA) to assess the microbial community structure in the soil and rhizosphere community of non-transgenic watermelons and transgenic watermelons in Miryang farmlands in Korea during the spring and summer of 2005. The PLFA data were seasonally examined for the number of PLFA to determine whether there is any difference in the microbial community in soils from two types of watermelons, non-transgenic and transgenic. We identified 78 PLFAs from the rhizosphere samples of the two types of watermelons. We found eight different PLFAs for the type of plants and sixteen PLFAs for the interaction of plant type and season. The PLFA data were analyzed by analysis of variance separated by plant type (P<0.0085), season (P<0.0154), and the plant type${\times}$season interaction (P<0.1595). Non-parametric multidimensional scaling (NMS showed a small apparent difference but multi-response permutation procedures (MRPP) confirmed that there was no difference in microbial community structure for soils of both plant types. Conclusively, there was no significant adverse effect of transgenic watermelon on bacterial and fungal relative abundance as measured by PLFA. We could reject our hypothesis that there might be an adverse effect from transgenic watermelon with our statistical results. Therefore, we can suggest the use of this PLFA methodology to examine the adverse effects of transgenic plants on the soil microbial community.

Effects of transgenic watermelon with CGMMV resistance on the diversity of soil microbial communities using PLFA

이훈복,김창기 한국통합생물학회 2010 Animal cells and systems Vol.14 No.3

We compared the composition of phospholipid fatty acids (PLFA) to assess the microbial community structure in the soil and rhizosphere community of non-transgenic watermelons and transgenic watermelons in Miryang farmlands in Korea during the spring and summer of 2005. The PLFA data were seasonally examined for the number of PLFA to determine whether there is any difference in the microbial community in soils from two types of watermelons, nontransgenic and transgenic. We identified 78 PLFAs from the rhizosphere samples of the two types of watermelons. We found eight different PLFAs for the type of plants and sixteen PLFAs for the interaction of plant type and season. The PLFA data were analyzed by analysis of variance separated by plant type (PB0.0085), season (PB0.0154), and the plant typeseason interaction (PB0.1595). Non-parametric multidimensional scaling (NMS)showed a small apparent difference but multi-response permutation procedures (MRPP) confirmed that there was no difference in microbial community structure for soils of both plant types. Conclusively, there was no significant adverse effect of transgenic watermelon on bacterial and fungal relative abundance as measured by PLFA. We could reject our hypothesis that there might be an adverse effect from transgenic watermelon with our statistical results. Therefore, we can suggest the use of this PLFA methodology to examine the adverse effects of transgenic plants on the soil microbial community.

Generation and molecular characterization of marker-free Bt transgenic rice plants by selectable marker-less transformation

우희종,이승범,Yang Qin,임명호,이진형,신공식,조현석,박순기 한국식물생명공학회 2015 Plant biotechnology reports Vol.9 No.6

Public concern about the safety of genetically modified (GM) crops and potential pleiotropic effects of selectable marker gene insertion and expression can be avoided by developing a selectable marker-free transformation system. Various techniques have been developed to produce marker-free transgenic plants; however, an approach that does not require the use of selectable marker genes would not require post-transformation processes. We report such a method for generating of GM rice plants. To develop transgenic rice resistant to lepidopteran insects, a marker-free binary vector harboring the Bacillus thuringiensis mcyr1Ac gene was constructed and used for Agrobacterium tumefaciens-mediated transformation of rice scutellar calli. Initial screening of putative T0 transgenic rice plants was performed using polymerase chain reaction (PCR) on pools of DNA from 15 to 30 regenerated shoots, followed by genomic DNA PCR and Cry1Ac ImmunoStrip assays of individual plant extracts. Although the overall rice transformation efficiency of the non-selection approach is lower than that of traditional rice transformation by using marker genes, we derived six independent homozygous T3 events from Cry+ T0 shoots. Transgenes were detected and inheritance confirmed using DNA PCR, RT-PCR, and Southern blot analysis, and insertion sites were characterized by the sequencing of DNA flanking the transgenes. The transgene expression of cry1Ac was also stable and effective against rice leaf folder at levels comparable to those of a transgenic plant that had been derived by selection. This method could be applied to other plant species with similar transformation efficiencies to create selectable-marker-free transgenic plants for research and commercial uses.