Transgenic Colonies Research Articles

Production of transgenic first filial puppies expressing mutated human amyloid precursor protein gene.

Propagation of transgenic animals by germline transmission using assisted reproductive technologies such as in vitro fertilization (IVF) is the most efficient way to produce transgenic colonies for biomedical research. The objective of this study was to generate transgenic puppies from a founder dog expressing the mutated human amyloid precursor protein (mhAPP) gene. Experiment I assessed the characteristics of the semen prepared by freshly diluted, swim-up, and Percoll gradient methods using a computer-assisted semen analyzer (CASA). Motile and progressively motile sperm counts were higher in the Percoll gradient samples (p < 0.05) than in the swim-up and freshly diluted samples. In Experiment II, a total of 59, 70, and 65 presumptive zygotes produced by fresh, Percoll gradient, and swim-up methods, respectively, were transferred to surrogates (5 for each group); the Percoll gradient (27.27%) and swim-up samples (14.29%) showed the highest blastocyst formation rates, while fresh diluted semen did not produce any blastocyst. Experiment III examined the full-term developmental ability of embryos. Among the 5 surrogates in the Percoll gradient group, one (20.0%) became pregnant; it had 4 (6.15%) sacs and delivered 4 (6.15%; 2 males and 2 females) live puppies. Among the 4 puppies, 2 (50.0%) were found to transmit the transgene on their nail and toe under GFP fluorescence. Furthermore, the integration and expression of the mhAPP transgene were examined in the umbilical cords of all the IVF-derived puppies, and the presence of the transgene was only observed in the GFP-positive puppies. Thus, semen prepared by the Percoll method could generate transgenic puppies by male germline transmission using the IVF technique. Our result will help propagate transgenic dogs efficiently, which will foster human biomedical research.

Determination and analysis of sex ratios in heme oxygenase 1 gene targeted mouse embryos.

Heme oxygenase 1 or Hmox1 enzyme is involved in catalyzing the first and rate-limiting step in heme breakdown reactions. Many studies have reported a partial lethality of Hmox1 knockout mice obtained from heterozygous breeding pairs. Similar results were obtained in our transgenic mice colony and a sex specific bias was observed in the favor of males in the adult mice. Hmox1 independent factors which could have caused this bias were initially analyzed and it was found that those factors were not a reason behind this anomaly. Certain studies involving gene knockout hinted toward a prenatal or neonatal lethality of female knockout mice embryos or pups, respectively. In order to check if this bias was occurring in embryonic stages, that is, either if mutant female embryos were dying or if heterozygous mothers were not carrying embryos to term, we analyzed the sex-ratios in mid- and late-gestational ages (9.5-13.5dpc and 14.5-18.5dpc, respectively). Our results did not indicate any significant difference in the sex ratios in embryonic stages; hence, it was concluded that females are not dying in embryonic stages. It can be speculated that these deaths were probably occurring at neonatal age. More studies are required to confirm that the lack of Hmox1 gene products is the sole reason for this female lethality.

Effective NPM1 plasmid standards selection for minimal/measurable residual disease monitoring in acute myeloid leukemia.

NPM1 plasmid standards are required for absolute quantification of minimal residual disease in acute myeloid leukemia patients. The standards are usually obtained, next to commercially constructed gene fragments, from transgenic bacteria colonies. However, this procedure is laborious and very time consuming. We have developed a PCR method that speeds up, simplifies, and streamlines the process of preparing NPM1 plasmid standards. The method is based on a combination of three primers, two surrounding the usual NPM1 mutation position and one over the mutation site. With this method, we were able to clearly distinguish plasmids with at least 15 different NPM1 mutations from the wild-type NPM1 plasmid. With the new approach, preparing NPM1 plasmid standards is easier, identifying NPM1-positive colonies is possible in less than a day and moreover, for a lower price than commercially constructed gene fragments.

Preparing an Insectary in Burkina Faso to Support Research in Genetic Technologies for Malaria Control.

The Institut de Recherche en Sciences de la Santé (IRSS) of Burkina Faso, West Africa, was the first African institution to import transgenic mosquitoes for research purposes. A shift from the culture of mosquito research to regulated biotechnology research and considerable management capacity is needed to set up and run the first insectary for transgenic insects in a country that applied and adapted the existing biosafety framework, first developed for genetically modified (GM) crops, to this new area of research. The additional demands arise from the separate regulatory framework for biotechnology, referencing the Cartagena Protocol on Biosafety, and the novelty of the research strain, making public understanding and acceptance early in the research pathway important. The IRSS team carried out extensive preparations following recommendations for containment of GM arthropods and invested efforts in local community engagement and training with scientific colleagues throughout the region. Record keeping beyond routine practice was established to maintain evidence related to regulatory requirements and risk assumptions. The National Biosafety Agency of Burkina Faso, Agence Nationale de Biosécurité (ANB), granted the permits for import of the self-limiting transgenic mosquito strain, which took place in November 2016, and for conducting studies in the IRSS facility in Bobo-Dioulasso. Compliance with permit terms and conditions of the permits and study protocols continued until the conclusion of studies, when the transgenic colonies were terminated. All this required close coordination between management and the insectary teams, as well as others. This article outlines the experiences of the IRSS to support others undertaking such studies. The IRSS is contributing to the ongoing development of genetic technologies for malaria control, as a partner of Target Malaria. The ultimate objective of the innovation is to reduce malaria transmission by using GM mosquitoes of the same species released to reduce the disease-vectoring native populations of Anopheles gambiae s.l.

Alpha-tubulin promoter from Chlorella vulgaris allows genetic transformation of green coccoid microalga

Microalgae have become a feasible platform for high-value recombinant protein production. Although diverse sets of genetic tools for microalgae transformation have been developed, some critical elements, such as endogenous promoters, need to be improved for increasing transgene expression levels after genetic transformation. This work aims to evaluate a sequence from the 5′ upstream region of the alpha-tubulin gene from Chlorella vulgarisas a promoter for the expression of an antibiotic resistant gene in Chlorella sorokiniana. Using in silicoanalysis it was possible to identify a proximal promoter corresponding to 281 nucleotides upstream of the ATG start codon, which possessed 9 potential cis-regulatory element, including TATA Box and CAAT Box. The proximal promoter sequencewas used to drive the expression of a streptomycin-resistance gene (aada), previous optimization of its codon usage. The codon optimization of the aadasequence allowed it to obtain a GC content of 69.8% (compared to 53% of the original sequence), which increases its similarity with Chlorellasp. genomes (67.2%). Both genetic elements were cloned into the pUC57-Kan vector and transformed into C. sorokiniana cells by electroporation. The microalgae transgenic colonies were identified through culture in selective medium and PCR. Our results proved the capacity of the Chlorella vulgaris alpha-tubulin promoter to express a foreign antibiotic-resistance gene in C. sorokinianacells. Research of endogenous promoting sequences is essential in order to accomplish an efficient heterologous gene expression, especially in microalgae used for industrial production like those from Chlorella genus.

A simple genotyping method to detect small CRISPR-Cas9 induced indels by agarose gel electrophoresis

CRISPR gene editing creates indels in targeted genes that are detected by genotyping. Separating PCR products generated from wild-type versus mutant alleles with small indels based on size is beyond the resolution capacity of regular agarose gel electrophoresis. To overcome this limitation, we developed a simple genotyping method that exploits the differential electrophoretic mobility of homoduplex versus heteroduplex DNA hybrids in high concentration agarose gels. First, the CRISPR target region is PCR amplified and homo- and hetero-duplexed amplicons formed during the last annealing cycle are separated by 4–6% agarose gel electrophoresis. WT/mutant heteroduplexes migrate more slowly and are distinguished from WT or mutant homoduplexes. Heterozygous alleles are immediately identified as they produce two distinct bands, while homozygous wild-type or mutant alleles yield a single band. To discriminate the latter, equal amounts of PCR products of homozygous samples are mixed with wild-type control samples, subjected to one denaturation/renaturation cycle and products are electrophoresed again. Samples from homozygous mutant alleles now produce two bands, while those from wild-type alleles yield single bands. This method is simple, fast and inexpensive and can identify indels >2 bp. in size in founder pups and genotype offspring in established transgenic mice colonies.

Maintaining Quality of Candidate Strains of Transgenic Mosquitoes for Studies in Containment Facilities in Disease Endemic Countries.

Transgenic mosquitoes are being developed as novel components of area-wide approaches to vector-borne disease control. Best practice is to develop these in phases, beginning with laboratory studies, before moving to field testing and inclusion in control programs, to ensure safety and prevent costly field testing of unsuitable strains. The process of identifying and developing good candidate strains requires maintenance of transgenic colonies over many generations in containment facilities. By working in disease endemic countries with target vector populations, laboratory strains may be developed and selected for properties that will enhance intended control efficacy in the next phase, while avoiding traits that introduce unnecessary risks. Candidate strains aiming toward field use must consistently achieve established performance criteria, throughout the process of scaling up from small study colonies to production of sufficient numbers for field testing and possible open release. Maintenance of a consistent quality can be demonstrated by a set of insect quality and insectary operating indicators, measured over time at predetermined intervals. These indicators: inform comparability of studies using various candidate strains at different times and locations; provide evidence of conformity relevant to compliance with terms of approval for regulated use; and can be used to validate some assumptions related to risk assessments covering the contained phase and for release into the environment.

Evaluation of Agrobacterium-mediated Transformation of Chlamydomonas reinhardtii using a Synthetic amorpha-4, 11-diene Synthase Gene

Amorpha-4,11-diene synthase (ADS) is a key enzyme in biochemical pathway of the antimalarial agent artemisinin. An Agrobacterium-mediated transformation was carried out to express a synthetic ADS gene in green microalga Chlamydomonas reinhardtii strain 125C with bacterial strains GV3101 and LBA4404. The foreign gene was optimized based on codon usage bias of the microalga. Integration of the ADS in nuclear genome of C. reinhardtii was confirmed by polymerase chain reaction assay. The transgenic colonies cultured on selective medium turned yellow after three days and gradually died. Transformation procedure, growth habit of the transgenic microalgae together with probable causes of transformants loss is discussed. The present study is the first investigation for production of ADS enzyme in a microalgal system.

Development of sheep kidney cells with increased resistance to different subgenotypes of BVDV-1 by RNA interference

Bovine viral diarrhea virus (BVDV) should be a ubiquitous viral pathogen to the cattle and sheep industry. This pathogen is responsible for severe economic losses. We previously showed that plasmid-mediated dual short hairpin RNA (shRNA) efficiently inhibit BVDV replication in bovine kidney epithelial (MDBK) cells. In this study, we delivered the dual shRNA system to sheep fibroblasts and generated transgenic cell colonies. These transgenic fibroblasts were further used for somatic cell nuclear transfer (SCNT). Three lambs were born at full term, but perished soon after birth. Integration of shRNA into the genome of cloned sheep was confirmed by PCR and expression of shRNA in transgenic sheep was confirmed by real-time PCR. Kidney epithelial cells were isolated from transgenic sheep and challenged with multiple BVDV subgenotypes (BVDV-1a, BVDV-1b and BVDV-1c). The dual shRNA expressed in transgenic kidney epithelial cells significantly inhibited BVDV replication in a cross-resistance manner. Our results showed that transgenic RNAi might be a useful tool for preparation of transgenic animals with increased resistance to BVDV.

Transgenic expression of human cytoxic T-lymphocyte associated antigen4-immunoglobulin (hCTLA4Ig) by porcine skin for xenogeneic skin grafting.

Porcine skin is frequently used as a substitute of human skin to cover large wounds in clinic practice of wound care. In our previous work, we found that transgenic expression of human cytoxicT-lymphocyte associated antigen4-immunoglobulin (hCTLA4Ig) in murine skin graft remarkably prolonged its survival in xenogeneic wounds without extensive immunosuppression in recipients, suggesting that transgenic hCTLA4Ig expression in skin graft may be an effective and safe method to prolong xenogeneic skin graft survival. In this work, using a transgene construct containing hCTLA4Ig coding sequence under the drive of human Keratine 14 (k14) promoter, hCTLA4Ig transgenic pigs were generated by somatic nuclear transfer. The derived transgenic pigs were healthy and exhibited no signs of susceptibility to infection. The hCTLA4Ig transgene was stably transmitted through germline over generations, and thereby a transgenic pig colony was established. In the derived transgenic pigs, hCTLA4Ig expression in skin was shown to be genetically stable over generations, and detected in heart, kidney and corneal as well as in skin. Transgenic hCTLA4Ig protein in pigs exhibited expected biological activity as it suppressed human lymphocyte proliferation in human mixed lymphocyte culture to extents comparable to those of commercially purchased purified hCTLA4Ig protein. In skin grafting from pigs to rats, transgenic porcine skin grafts exhibited remarkably prolonged survival compared to the wild-type skin grafts derived from the same pig strain (13.33 ± 3.64 vs. 6.25 ± 2.49 days, P < 0.01), further indicating that the transgenic hCTLA4Ig protein was biologically active and capable of extending porcine skin graft survival in xenogeneic wounds. The transgenic pigs generated in this work can be used as a reproducible resource to provide porcine skin grafts with extended survival for wound coverage, and also as donors to investigate the impacts of hCTLA4Ig on xenotransplantation of other organs (heart, kidney and corneal) due to the ectopic transgenic hCTLA4Ig expression.

Large-scale production of functional human lysozyme in transgenic cloned goats

Human lysozyme (hLZ), an essential protein against many types of microorganisms, has been expressed in transgenic livestock to improve their health status and milk quality. However, the large-scale production of hLZ in transgenic livestock is currently unavailable. Here we describe the generation of transgenic goats, by somatic cell-mediated transgenic cloning, that express large amounts of recombinant human lysozyme (rhLZ) in milk. Specifically, two optimized lysozyme expression cassettes (β-casein/hLZ and β-lactoglobulin/hLZ) were designed and introduced into goat somatic cells by cell transfection. Using transgenic cell colonies, which were screened by 0.8mg/mL G418, as a nuclear donor, we obtained 10 transgenic cloned goats containing one copy of hLZ hybrid gene. An ELISA assay indicated that the transgenic goats secreted up to 6.2g/L of rhLZ in their milk during the natural lactation period, which is approximately 5–10 times higher than human milk. The average rhLZ expression levels in β-casein/hLZ and β-lactoglobulin/hLZ transgenic goats were 2.3g/L and 3.6g/L, respectively. Therefore, both rhLZ expression cassettes could induce high levels of expression of the rhLZ in goat mammary glands. In addition, the rhLZ purified from goat milk has similar physicochemical properties as the natural human lysozyme, including the molecular mass, N-terminal sequence, lytic activity, and thermal and pH stability. An antibacterial analysis revealed that rhLZ and hLZ were equally effective in two bacterial inhibition experiments using Staphylococcus aureus and Escherichia coli. Taken together, our experiments not only underlined that the large-scale production of biologically active rhLZ in animal mammary gland is realistic, but also demonstrated that rhLZ purified from goat milk will be potentially useful in biopharmaceuticals.

A Transgenic Tri-Modality Reporter Mouse

Transgenic mouse with a stably integrated reporter gene(s) can be a valuable resource for obtaining uniformly labeled stem cells, tissues, and organs for various applications. We have generated a transgenic mouse model that ubiquitously expresses a tri-fusion reporter gene (fluc2-tdTomato-ttk) driven by a constitutive chicken β-actin promoter. This “Tri-Modality Reporter Mouse” system allows one to isolate most cells from this donor mouse and image them for bioluminescent (fluc2), fluorescent (tdTomato), and positron emission tomography (PET) (ttk) modalities. Transgenic colonies with different levels of tri-fusion reporter gene expression showed a linear correlation between all three-reporter proteins (R2=0.89 for TdTomato vs Fluc, R2=0.94 for Fluc vs TTK, R2=0.89 for TdTomato vs TTK) in vitro from tissue lysates and in vivo by optical and PET imaging. Mesenchymal stem cells (MSCs) isolated from this transgenics showed high level of reporter gene expression, which linearly correlated with the cell numbers (R2=0.99 for bioluminescence imaging (BLI)). Both BLI (R2=0.93) and micro-PET (R2=0.94) imaging of the subcutaneous implants of Tri-Modality Reporter Mouse derived MSCs in nude mice showed linear correlation with the cell numbers and across different imaging modalities (R2=0.97). Serial imaging of MSCs transplanted to mice with acute myocardial infarction (MI) by intramyocardial injection exhibited significantly higher signals in MI heart at days 2, 3, 4, and 7 (p<0.01). MSCs transplanted to the ischemic hindlimb of nude mice showed significantly higher BLI and PET signals in the first 2 weeks that dropped by 4th week due to poor cell survival. However, laser Doppler perfusion imaging revealed that blood circulation in the ischemic limb was significantly improved in the MSCs transplantation group compared with the control group. In summary, this mouse can be used as a source of donor cells and organs in various research areas such as stem cell research, tissue engineering research, and organ transplantation.

Medical applications of RNA interference (RNAi)

During its long and rich history, medical sciences have undergone several changes of paradigms. In this quest to develop more potent drugs, less invasive surgical techniques and more sophisticated diagnostic approaches (among other goals) medicine has contributed decisively to our well being and longevity. Some of these major accomplishments, which altered the course of humanity are: the development of antibiotics, organ transplantation and radiography. Medicine has recently reached a new hallmark: RNA interference (RNAi), a Nobel prize winning technology which promises to promote medical care at the molecular level, by regulating genes in our favor. RNAi was first successfully used in C. elegans, a laboratory model species, by microinjecting dsRNAs in the worm’s gonad. However, the works that laid the foundations for the development of the technique were initially done in plants and fungi. RNAi was accidentally discovered, when white transgenic petunias were obtained instead of deep purple ones [1]; a similar paradoxal result was obtained when white transgenic colonies of Neurospora sp were observed in place of orange ones [2]. These findings revealed the existence of an unknown potent mechanism of gene regulation, which could be tamed for diverse uses, from basic science, agriculture, animal health and medicine. From these seminal observations, during a period of nearly 10 years, researchers aimed at characterizing, at the genetic and biochemical levels, the process of RNAi. By the year 2001, scientists were ready to perform the initial analyses in human cells [3]. Since then, RNAi has been used as a tool in molecular biology, allowing potent and specific gene silencing. This is achieved by the introduction of double-stranded RNA molecules (dsRNAs), which ultimately lead to the degradation of messenger RNAs (mRNAs), i.e., gene silencing. The molecular mechanism behind RNAi requires the activation of an endogenous pathway which is present in almost all eukaryotic cells. This pathway is responsible for detecting long dsRNAs and, subsequently, promoting their cleavage by an enzyme called dicer, into small duplexes of approximately 21-25 bp called siRNAs (small interfering RNAs). Alternatively, siRNAs can be directly introduced into the cytoplasm of eukaryotic cells. This second approach is required in mammalian cells, since dsRNAs longer than 30 bp induce apoptosis. siRNAs are subsequently transferred to a complex called RISC (RNA-induced silencing complex), where one strand of the duplex is destroyed and the other remains intact (designated guide strand). RISC then searches the cytoplasm for mRNA molecules with sequence complementarity to the guide strand. Once a mRNA with full complementarity is found, slicer (an endoribonuclease which is part of RISC) performs the cleavage of target mRNA [4]. Therefore, RNAi can be used as a technique that takes advantage of an endogenous biochemical pathway, directing it to the destruction of specific target transcripts (gene silencing). In addition, RNAi can be used to produce transgenic animals (known as knockdown animals), resembling the null phenotype (- / -). Additionally, since RNAi may be tamed to promote silencing efficiencies ranging from 0.1% to 100% [5,6], animals with partial phenotypes , known as hypomorfic, may generated. These hypomorfic individuals are very informative since they reveal intermediary and unprecedented phenotypes and because the null genetic construction may happen to be unviable [7]. Currently, most studies use RNAi as a tool for reverse genetics (identification of gene function), but the applications are numerous: i) disease control (viruses [8]; bacterial diseases [9]; parasites, [10]); genetic [11]; tumors [12], ii) production of animals of commercial interest [13] and iii) production of animal models for research use [14]. Other possible future applications include: control of drug consumption [15], pain relief [16], modulation of sleep [17], among many others. Another relevant feature of RNAi is its temporary effect - long lasting knockdown is only observed in C. elegans and a few other species. Therefore, in order to study genes with constitutive expression, it is necessary to combine long-term strategies such as the production of transgenic animals encoding shRNAs (similar to siRNAs) or the use of viral vectors. On the other hand, this transitory characteristic can also be used as an advantage, when one considers RNAi as a therapetical strategy. Furthermore, the control of gene expression via RNAi can be modulated in time and directed to specific cells or organs. The identification of viral proteins capable of suppressing RNAi, such as p19 [18] allows for future applications, as well as the use of inducible promoters (high temperature, drugs). All these new developments have recently led to the first published human clinical trials with very promising results [19].

234 EFFICIENT EXPRESSION OF HUMAN ENDOTHELIAL PROTEIN C RECEPTOR AND HUMAN THROMBOMODULIN IN TRANSFECTED PIG PRIMARY hCD55+-GAL–/– FIBROBLASTS USING F2A EXPRESSION VECTOR

The genetic engineering of the pig genome for xenotransplantation studies requires the insertion of different transgenes to create multi-transgenic pigs. In order to simultaneously add more transgene in a single genetic insertion, we constructed a polycistronic vector using the F2A self-cleaving peptide. Moreover, this solution has the added advantages of preventing possible segregation during breeding of the animals and of guaranteeing an equimolar production of chosen transgenes. The scope of this work was the construction and validation of an ubiquitous F2A-bicistronic expression vector for human thrombomodulin (hTM) and human endothelial protein C receptor (hEPCR) genes in pig primary hCD55-GAL–/– cells to establish transgenic fibroblasts colonies, to be used for somatic cell nuclear transfer (SCNT) to generate pigs for xenotransplantation research. The expression vector consisted of pCAGGS promoter (CMV-IE+chicken β actin) followed by hEPCR-furinF2A-hTM coding sequence. The resulting expression cassette was inserted between 2 insulators obtained from the 5′ MAR region of chicken lysozyme. Outside of this insulated structure, there is a loxable puromycin selection cassette. The resulting purified and linearized expression vector (pEFTM/Lgu I = 5 μg) was transfected into hCD55-GAL–/– primary fibroblasts (1 × 106), using Nucleofector (Amaxa, Lonza, Cologne, Germany), in parallel for comparative purposes we cotransfected the 2 pCAGGS-monocistronic vectors for the same transgenes (hEPCR and hTM = 1:3, 5 μg). Transfected cells were selected with puromycin (1 μg mL–1) for 15 days. After 8 days of selection, resistant colonies were picked up and expanded into 24-well plates for cryopreservation and analyses. Bicistronic transfection produced 20 clones and cotransfection only 8 clones that were analysed by Western blot (WB) and by immunocytochemistry (ICC) using polyclonal antibody anti-EPCR (1:250, R&amp;D) and monoclonal antibody ab6980-Abcam (1:5000, Abcam, Cambridge, UK) in WB; polyclonal antibody RCR252 (1:100, Sigma-Aldrich, St. Louis, MO, USA) and monoclonal antibody ab6980-Abcam (1:100, Abcam) for ICC. Seventeen bicistronic clones (85%) and 2 cotransfected monocistronic clones (25%) were positive for both transgenes using WB. After ICC analyses, only 11 bicistronic colonies (55%) and 1 cotransfected colony (12.5%) uniformly expressed the desired transgenes and were selected for SCNT. The pCAGGS promoter maintained its strong expression also using the hEPCR-FurinF2A-hTM coding sequence and this bicistronic solution permitted us to improve our results obtained with co-transfection. Availability of hEPCR+ hTM+ hCD55+-GAL–/– colonies will allow us to obtain a new transgenic background for future xenotransplantation projects. This study was supported by EU grant no. LSHB-CT-2006-037377 (Xenome) and by Regione Lombardia (Superpig project).

Non‐invasive in vivo imaging for liver tumour progression using an orthotopic hepatocellular carcinoma model in immunocompetent mice

Maintenance of complex transgenic colonies and labour-intensive techniques pose significant challenges in work involving mouse models for hepatocellular carcinoma (HCC). Other animal models of unusual species are generally impractical for research purposes. To develop a highly reproducible orthotopic mouse model for HCC based on the murine α-foetoprotein (AFP), producing cell line Hepa1-6 and to monitor liver tumour progression via in vivo imaging, and measurement of plasma AFP. Intrahepatic tumour was induced following subcapsular implantation of 10(+6) Hepa1-6 cells into C57L/J mice. AFP production was examined in vitro and in vivo using immunoblotting. Three confirmatory non-invasive imaging modalities were applied to follow tumour progression over time including ultrasound biomicroscopy (UBM), micromagnetic resonance imaging (microMRI), and bioluminescence. α-foetoprotein expression was confirmed both in vitro and in vivo, with increasing levels in the plasma as tumours progressed. UBM, microMRI and bioluminescence detected intrahepatic tumours to a 2 mm resolution by day 14. Sequential imaging studies demonstrated an intrahepatic pattern of disease progression with an observed median survival of 29 days. Immunosuppression of tumour-bearing mice led to a greater tumour size and decreased survival. Intrahepatic implantation of Hepa1-6 as a mouse model for HCC is a highly reproducible in vivo system with tumour biology analogous to human disease and is regulated by the presence of an intact host immune system. Tumour progression may be monitored in vivo by UBM, microMRI and bioluminescence. Plasma AFP increases over time, allowing redundancy in non-invasive means of following tumour progression.

Expression of organophosphorus-degradation gene (opd) in aggregating and non-aggregating filamentous nitrogen-fixing cyanobacteria

Genetic engineering in filamentous N2-fixing cyanobacteria usually involves Anabaena sp. PCC 7120 and several other non-aggregating species. Mass culture and harvest of such species are more energy consuming relative to aggregating species. To establish a gene transfer system for aggregating species, we tested many species of Anabaena and Nostoc, and identified Nostoc muscorum FACHB244 as a species that can be genetically manipulated using the conjugative gene transfer system. To promote biodegradation of organophosphorus pollutants in aquatic environments, we introduced a plasmid containing the organophosphorus-degradation gene (opd) into Anabaena sp. PCC 7120 and Nostoc muscorum FACHB244 by conjugation. The opd gene was driven by a strong promoter, PpsbA. From both species, we obtained transgenic strains having organophosphorus-degradation activities. At 25°C, the whole-cell activities of the transgenic Anabaena and Nostoc strains were 0.163±0.001 and 0.289±0.042 unit/μg Chl a, respectively. However, most colonies resulting from the gene transfer showed no activity. PCR and DNA sequencing revealed deletions or rearrangements in the plasmid in some of the colonies. Expression of the green fluorescent protein gene from the same promoter in Anabaena sp. PCC 7120 showed similar results. These results suggest that there is the potential to promote the degradation of organophosphorus pollutants with transgenic cyanobacteria and that selection of high-expression transgenic colonies is important for genetic engineering of Anabaena and Nostoc species. For the first time, we established a gene transfer and expression system in an aggregating filamentous N2-fixing cyanobacterium. The genetic manipulation system of Nostoc muscorum FACHB244 could be utilized in the elimination of pollutants and large-scale production of valuable proteins or metabolites.

BioSpotlight

BioSpotlight

Isolation of Transfected Adult Fibroblast Clones and SCNT Embryo Research*

Transgenesis in rabbit has provided numerous opportunities for livestock production. The development of nuclear transfer (NT) technology has improved the production of transgenic livestock by the combination of transgenic technology. However, the isolation of pure colonies from a single transfection event remains laborious and can be a constraint in the production of transgenic livestock. 24-well cell culture plates were used to isolate cell lineages obtained from a single fibroblast clone transfected with the pRNAT-U6.1/Neo plasmid. Since single fibroblast clone does not grow well in fresh medium, the use of conditioned medium was evaluated. Meanwhile, the effect of initial cell density and linear-plasimid on transgenic fibroblast colony growth were investigated. The increasing initial cell density and lining plasmid could improve colony growth and expansion. There was a significant difference in the conditioned medium or initial cell density compared to the control group. The neomycin phosphotransferase gene was detected in isolated colonies and NT embryos were produced from these cells. When the transgenic fibroblasts were used as donor cells of nuclear transfer, the blastocyst rate were 23.5%. There was not a significant difference in the transgenic fibroblasts compared to the normal group. PCR or Multiplex-PCR assays were performed to detect the transfected fragment as well as autosomal β-actin DNA in single NT embryos. This approach provided a reliable method for isolating transfected mammalian cells and for diagnosing the incorporation of desirable vectors in NT embryos. This method can reduce the time and cost of transgenic livestock production; further improvements in related technologies will facilitate the use of this method for the generation of the genetic engineering of rabbits.

Optimization of square-wave electroporation for transfection of porcine fetal fibroblasts

Development of a transgenic porcine biomedical research model requires effective delivery of DNA into the donor cell followed by selection of genetically modified somatic cell lines to be used for nuclear transfer. The objective of the current study was 2-fold: (1) to compare the effectiveness of a single 1 ms pulse of different voltages (V; 100, 150, 200, 250, 300, 350) and multiple 1 ms pulses (1, 2, 3, 4 or 5) at 300 V for delivery and expression of super-coiled GFP vector in surviving cells of three fetal fibroblast cell lines, and (2) to determine the ability of these electroporation parameters to produce stably transfected fibroblast colonies following G418 selection. Cell line (P < 0.001) and voltage (P < 0.001) affected DNA delivery into the cell as assessed by GFP expression while survival at 24 h was affected by voltage (P < 0.001) and not by cell line (P = 0.797). Using a single pulse while increasing voltage resulted in the percentage of GFP expressing cells increasing from 3.2 +/- 0.8% to 43.0 +/- 3.4% while survival decreased from 90.5 +/- 8.0% to 44.8 +/- 2.0%. The number of pulses at 300 V significantly affected survival (P < 0.001) and GFP expression (P < 0.001). Survival steadily decreased following 1-5 pulses from 63.2 +/- 6.3% to 3.0 +/- 0.3% with GFP expression of surviving cells increasing from 35.6 +/- 2.67% to 71.4 +/- 6.1%. Electroporation of a selectable marker at a 1:1 copy number ratio to a co-electroporated transgene resulted in 83% of G418 resistant colonies also being PCR positive for the secondary transgene. These electroporation conditions, specifically, three 1 ms pulses of 300 V to 200 muL of 1 x 10(6) cells/mL in the presence of 12.5 mug DNA/mL effectively introduced DNA into somatic cells. The utilization of these conditions produced numerous transgenic fibroblast colonies following G418 selection that when used for somatic cell nuclear transfer resulted in the production of live offspring.

“What’s wrong with my monkey?” Ethical perspectives on germline transgenesis in marmosets

The birth of the first transgenic primate to have inherited a transgene from its parents opens the possibility to set up transgenic marmoset colonies, as these monkeys are small and relatively easy to keep and breed in research facilities. The prospect of transgenic marmoset models of human disease, readily available in the way that transgenic laboratory mice are currently, prompts excitement in the scientific community; but the idea of monkeys being bred to carry diseases is also contentious. We structure an ethical analysis of the transgenic marmoset case around three questions: whether it is acceptable to use animals as models of human disease; whether it is acceptable to genetically modify animals; and whether these animals' being monkeys makes a difference. The analysis considers the prospect of transgenic marmoset studies coming to replace transgenic mouse studies and lesion studies in marmosets in some areas of research. The mainstream, broadly utilitarian view of animal research suggests that such a transition will not give rise to greater ethical problems than those presently faced. It can be argued that using marmosets rather than mice will not result in more animal suffering, and that the benefits of research will improve with a move to a species more similar in phylogenetic terms to humans. The biological and social proximity of monkeys and humans may also benefit the animals by making it easier for scientists and caretakers to recognize signs of suffering and increasing the human motivation to limit it. The animal welfare and research impacts of the transition to marmoset use will depend very much on the extent to which researchers take these issues seriously and seek to minimize animal harm and optimize human benefit.