Transgenesis System Research Articles

Transposon-mediated chromosomal integration of transgenes in the parasitic nematode Strongyloides ratti and establishment of stable transgenic lines.

Genetic transformation is a potential tool for analyzing gene function and thereby identifying new drug and vaccine targets in parasitic nematodes, which adversely affect more than one billion people. We have previously developed a robust system for transgenesis in Strongyloides spp. using gonadal microinjection for gene transfer. In this system, transgenes are expressed in promoter-regulated fashion in the F1 but are silenced in subsequent generations, presumably because of their location in repetitive episomal arrays. To counteract this silencing, we explored transposon-mediated chromosomal integration of transgenes in S. ratti. To this end, we constructed a donor vector encoding green fluorescent protein (GFP) under the control of the Ss-act-2 promoter with flanking inverted tandem repeats specific for the piggyBac transposon. In three experiments, free-living Strongyloides ratti females were transformed with this donor vector and a helper plasmid encoding the piggyBac transposase. A mean of 7.9% of F1 larvae were GFP-positive. We inoculated rats with GFP-positive F1 infective larvae, and 0.5% of 6014 F2 individuals resulting from this host passage were GFP-positive. We cultured GFP-positive F2 individuals to produce GFP-positive F3 L3i for additional rounds of host and culture passage. Mean GFP expression frequencies in subsequent generations were 15.6% in the F3, 99.0% in the F4, 82.4% in the F5 and 98.7% in the F6. The resulting transgenic lines now have virtually uniform GFP expression among all progeny after at least 10 generations of passage. Chromosomal integration of the reporter transgenes was confirmed by Southern blotting and splinkerette PCR, which revealed the transgene flanked by S. ratti genomic sequences corresponding to five discrete integration sites. BLAST searches of flanking sequences against the S. ratti genome revealed integrations in five contigs. This result provides the basis for two powerful functional genomic tools in S. ratti: heritable transgenesis and insertional mutagenesis.

3 MEGANUCLEASE TRANSGENESIS IN IVF AND CLONED BOVINE PREIMPLANTATORY EMBRYOS

Techniques available for production of transgenic animals remain inefficient. Recently an effective transgenesis system using I-SceI meganuclease (MegaN) has been described in Xenopus. It consists of MegaN incubation with a pIS-plasmid carrying I-SceI recognition sites-, followed by injection into fertilized egg. Here, we evaluated the MegaN transgenesis system combined with new transgenesis approaches developed by our group. We compared (1) three pIS concentrations incubated or not with MegaN; (2) injection of oocytes at germinal vesicle stage (GV) or of presumptive zygotes post IVF; (3) cloning using donor cells briefly incubated with MegaN or with pIS alone. Conditions for MegaN digestion were 0.5 UI I-SceI + 1x I-SceI buffer +pIS, at 37°C for 45 min. Two pIS were tested. For #1, IS- PaxGFP at 15, 25 and 50 ng μL–1 (15+, 25+ and 50+ for transgene digested by MegaN and 15-, 25- and 50- for transgene not digested). For #2, 50 ng μL–1 of IS-CXEGFP was injected with or without MegaN at GV or after IVF (EGFP+GV, EGFP–GV, EGFP+IVF, EGFP–IVF). Finally, IS-CXEGFP digested or not by MegaN (EGFP+ or EGFP–) and incubated with cumulus cells for 5 min were intracytoplasmatically injected into enucleated oocytes. Embryos were cultured in SOF. Rates of blastocysts and egfp expression were evaluated. Data was analysed by Fisher's test (P < 0.05). In Experiment 1, no differences in blastocyst rates were observed between the 3 IS-PAXGFP concentrations; however, except for 25+ and 50+, blastocysts rates were lower than after IVF [13/69 (18%), 22/98 (22%), 25/89 (28%), 19/91 (20%), 22/80 (27%), 16/71 (22%) and 31/80 (38%) for 15+, 15–, 25+, 25–, 50+, 50– and IVF, respectively]. Egfp expression was higher for 50– than for other groups, except for 25–, which did not statistically differ [9/69 (13%), 12/98 (12%), 19/89 (21%), 24/91 (26%), 16/80 (20%) and 27/71 (38) for 15+, 15–, 25+, 25–, 50+ and 50–, respectively). We observed higher cleavage and blastocyst rates after injection post IVF as compared with GV (P < 0.05). Egfp expression rates were also higher after injection post IVF, but no differences were found between digestion or not with MegaN [19/95 (20%), 23/95 (24%), 79/115 (68%) and 69/119 (57%) for EGFP+GV, EGFP–GV, EGFP+IVF and EGFP–IVF, respectively]. Egfp blastocyst over total blastocyst rates did not differ between injection at GV or post IVF [3/4 (75%), 2/3 (66%), 32/40 (80%) and 35/46 (76%) for EGFP+GV, EGFP–GV, EGFP+IVF and EGFP–IVF, respectively]. After cloning, no differences were found between EGFP+ and EGFP– in blastocysts [10/57 (17%) and 13/70 (18%) for EGFP+ or EGFP–], or in Day 4 egfp expression (23/57 (40%) and 24/70 (34%), respectively]. Expression of blastocysts after cloning was high [9/10 (90%) and 10/13 (76%) for EGFP+ and EGFP–, respectively]. In conclusion, MegaN transgenesis can produce bovine transgene-expressing embryos, although no differences were observed between digestion or not with MegaN. Injection at GV resulted in transgene-expressing embryos. Cloning with donor cells briefly incubated with MegaN resulted in the highest transgenesis rates. MegaN transgenesis could be useful for transgenic live mammal production, as observed in Xenopus.

Novel approaches to in vitro transgenesis

The study of gene expression is a major focus in biological research and is recognised to be critical for our understanding of physiological and pathophysiological processes. Methods to study gene expression range from in vitro biochemical assays through cultured cells and tissue biopsies to whole organisms. In the early stages of project development, considerations about which model system to use should be addressed and may influence future experimental procedures. The aim of this review is to briefly describe advantages and disadvantages of the existing techniques available to study eukaryote gene expression in vitro, including the mechanism of transgene integration (transient or stable), the different transgenesis systems available, including plasmids, viruses and targeted integration and knockin approaches, and paying particular attention to expression systems such as bacterial artificial chromosomes and episomal vectors that offer a number of advantages and are increasing in popularity. We also discuss novel approaches that combine some of the above techniques, generating increasingly complex but physiologically accurate expression systems.

Pronuclear injection-based mouse targeted transgenesis for reproducible and highly efficient transgene expression

Mouse transgenesis has proven invaluable for analysis of gene function and generation of human disease models. We describe here the development of a pronuclear injection-based targeted transgenesis (PITT) system, involving site-specific integration in fertilized eggs. The system was applied to two different genomic target loci to generate a series of transgenic lines including fluorescent mice, which reproducibly displayed strong, ubiquitous and stable transgene expression. We also demonstrated that knockdown mice could be readily generated by PITT by taking advantage of the reproducible and highly efficient expression system. The PITT system, which circumvents the problem of unpredictable and unstable transgene expression of conventional random-integration transgenic mice, reduces the time, cost and effort needed to generate transgenic mice, and is potentially applicable to both in vivo ‘gain-of-function’ and ‘loss-of-function’ studies.

A combination of transposable elements and magnetic cell sorting provides a very efficient transgenesis system for chicken primary erythroid progenitors

BackgroundStable transgenesis is an undeniable key to understanding any genetic system. Retrovirus-based insertional strategies, which feature several technical challenges when they are used, are often limited to one particular species, and even sometimes to a particular cell type as the infection depends on certain cellular receptors. A universal-like system, which would allow both stable transgene expression independent of the cell type and an efficient sorting of transfected cells, is required when handling cellular models that are incompatible with retroviral strategies.ResultsWe report here on the combination of a stable insertional transgenesis technique, based on the Tol2 transposon system together with the magnetic cell sorting (MACS) technique, which allows specific selection of cells carrying the transgene in an efficient, reliable and rapid way.ConclusionThis new Tol2/MACS system leads to stable expression in a culture of primary chicken erythroid cells highly enriched in cells expressing the transgene of interest. This system could be used in a wide variety of vertebrate species.

Keeping it simple: an easy method for manipulating the expression levels of malaria proteins

The ability to genetically manipulate malaria parasites in recent times has contributed considerably to our understanding of the biology of this deadly pathogen. Epp et al. have now expanded the repertoire of molecular tools available for the transgenesis system for the human malaria parasite Plasmodium falciparum by developing a simple methodology to regulate malaria gene expression. In this article, we comment on this technique and discuss its potential applications in the study of the biology of malaria parasites.

Transgenesis and neuronal ablation in parasitic nematodes: revolutionary new tools to dissect host–parasite interactions

Ease of experimental gene transfer into viral and prokaryotic pathogens has made transgenesis a powerful tool for investigating the interactions of these pathogens with the host immune system. Recent advances have made this approach feasible for more complex protozoan parasites. By contrast, the lack of a system for heritable transgenesis in parasitic nematodes has hampered progress toward understanding the development of nematode-specific cellular responses. Recently, however, significant strides towards such a system have been made in several parasitic nematodes, and the possible applications of these in immunological research should now be contemplated. In addition, methods for targeted cell ablation have been successfully adapted from Caenorhabditis elegans methodology and applied to studies of neurobiology and behaviour in Strongyloides stercoralis. Together, these new technical developments offer exciting new tools to interrogate multiple aspects of the host-parasite interaction following nematode infection.

A strong constitutive gene expression system derived from ibAGP1 promoter and its transit peptide

To develop a strong constitutive gene expression system, the activities of ibAGP1 promoter and its transit peptide were investigated using transgenic Arabidopsis and a GUS reporter gene. The ibAGP1 promoter directed GUS expression in almost entire tissues including rosette leaf, inflorescence stem, inflorescence, cauline leaf and root, suggesting that the ibAGP1 promoter is a constitutive promoter. GUS expression mediated by ibAGP1 promoter was weaker than that by CaMV35S promoter in all tissue types, but when GUS protein was targeted to plastids with the aid of the ibAGP1 transit peptide, GUS levels increased to higher levels in lamina, petiole and cauline leaf compared to those produced by CaMV35S promoter. The enhancing effect of ibAGP1 transit peptide on the accumulation of foreign protein was tissue-specific; accumulation was high in lamina and inflorescence, but low in root and primary inflorescence stem. The transit peptide effect in the leaves was maintained highly regardless of developmental stages of plants. The ibAGP1 promoter and its transit peptide also directed strong GUS gene expression in transiently expressed tobacco leaves. These results suggest that the ibAGP1 promoter and its transit peptide are a strong constitutive foreign gene expression system for transgenesis of dicot plants.

Antagonizing inactivated tumor suppressor genes and activated oncogenes by a versatile transgenesis system: application in mantle cell lymphoma

A broad range of malignant diseases, such as mantle cell lymphoma (MCL), is associated with complex genomic alterations, demanding multimodal functional testing of candidate genes. To assess such candidate disease genes, we have developed a bidirectional targeted transgenesis tool, which allows well-controlled modulation of individual gene activities within a cellular MCL system. The engineered versatile transgenesis system permits functional analysis of virtually any candidate gene: for tumor suppressor genes by complementation via integration of respective genomic DNA or for oncogenes by inactivation via integrated shRNA coding plasmids. Complementation by genomic DNA ensures wild-type (WT) regulated gene expression, whereas genomic integration of shRNA coding inserts by an advanced RNAi-strategy mediates specific knock-down of gene expression. Site-specific genomic integration of an unmodified BAC, which contains the CDKN2A/B genes absent in the MCL model system, restored CDKN2A/B expression resulting in the inhibition of cell proliferation. CCND1, strongly overexpressed in the model system, was down-regulated via shRNA expression, again inhibiting proliferation. Notably, the presented site-specific shRNA-strategy circumvents interference by IFN-response induced when using other RNAi gene knock-down methods. In conclusion, we here demonstrate that adequate restoration of a range of different gene activities yields in a desired antiproliferative effect in MCL-derived cells. By antagonizing inactivated tumor suppressor genes or activated oncogenes, the presented approach can be readily used for the functional analysis of a broad range of disease-related genetic defects.

Schistosoma mansoni miracidia transformed by particle bombardment infect Biomphalaria glabrata snails and develop into transgenic sporocysts

Miracidia (and adults) of Schistosoma mansoni which had been subjected to particle bombardment with a plasmid DNA encoding enhanced green fluorescent protein (EGFP) under control of the S. mansoni heat shock protein 70 (HSP70) promoter and termination elements were shown to express the reporter gene. Bombarded miracidia were able to penetrate and establish in Biomphalaria glabrata the intermediate host snail. Gold particles could be detected in the germ balls of parasites in paraffin-sections of snail tissue. The bombarded miracidia were able to develop normally and to transform into mother sporocysts. Reporter gene activity could be determined at 10 days post-infection by RT-PCR in snail tissues, but not by microscopy or Western blot which probably reflected sub-optimal expression levels of constructs. Our findings indicated that it is feasible to return transgenic miracidia to the life cycle, a crucial step for the establishment of a transgenesis system for schistosomes. Index Descriptors and Abbreviations: schistosome, miracidium, sporocyst, particle bombardment, gold particles, transgene, Biomphalaria glabrata; germ ball; EGFP, enhanced green fluorescent protein; RT-PCR, reverse transcription-polymerase chain reaction; gDNA, genomic DNA; PBS, phosphate buffered saline; TBS, Tris buffered saline; FCS, fetal calf serum; DEAE, diethylaminoethyldextran

Dominance of virus over host factors in cross-species activation of human cytomegalovirus early gene expression.

Human cytomegalovirus (HCMV) exhibits a highly restricted host range. In this study, we sought to examine the relative significance of host and viral factors in activating early gene expression of the HCMV UL54 (DNA polymerase) promoter in murine cells. Appropriate activation of the UL54 promoter at early times is essential for viral DNA replication. To study how the HCMV UL54 promoter is activated in murine cells, a transgenesis system based on yeast artificial chromosomes (YACs) was established for HCMV. A 178-kb YAC, containing a subgenomic fragment of HCMV encompassing the majority of the unique long (UL) region, was constructed by homologous recombination in yeast. This HCMV YAC backbone is defective for viral growth and lacks the major immediate-early (IE) gene region, thus permitting the analysis of essential cis-acting sequences when complemented in trans. To quantitatively measure the level of gene expression, we generated HCMV YACs containing a luciferase reporter gene inserted downstream of either the UL54 promoter or, as a control for late gene expression, the UL86 promoter, which directs expression of the major capsid protein. To determine the early gene activation pathway, point mutations were introduced into the inverted repeat 1 (IR1) element of the UL54 promoter of the HCMV YAC. In the transgenesis experiments, HCMV YACs and derivatives generated in yeast were introduced into NIH 3T3 murine cells by polyethylene glycol-mediated fusion. We found that infection of YAC, but not plasmid, transgenic lines with HCMV was sufficient to fully recapitulate the UL54 expression program at early times of infection, indicating the importance of remote regulatory elements in influencing regulation of the UL54 promoter. Moreover, YACs containing a mutant IR1 in the UL54 promoter led to reduced ( approximately 30-fold) reporter gene expression levels, indicating that HCMV major IE gene activation of the UL54 promoter is fully permissive in murine cells. In comparison with HCMV, infection of YAC transgenic NIH 3T3 lines with murine cytomegalovirus (MCMV) resulted in lower (more than one order of magnitude) efficiency in activating UL54 early gene expression. MCMV is therefore not able to fully activate HCMV early gene expression, indicating the significance of virus over host determinants in the cross-species activation of key early gene promoters. Finally, these studies show that YAC transgenesis can be a useful tool in functional analysis of viral proteins and control of gene expression for large viral genomes.

Isogenic transgenic homozygous fish induced by artificial parthenogenesis.

As a model system for vertebrate transgenesis, fish have many attractive advantages, especially with respect to the characteristics of eggs, allowing us to produce isogenic, transgenic, homozygous vertebrates by combining with chromosome-set manipulation. Here, we describe the large-scale production of isogenic transgenic homozygous animals using our experimental organism, the mud loach Misgurnus mizolepis, by the simple process of artificial parthenogenesis in a single generation. These isogenic fish have retained transgenic homozygous status in a stable manner during the subsequent 5 years, and exhibited increased levels of transgene expression. Furthermore, their isogenic nature was confirmed by cloned transgenic homozygous offspring produced via another step of parthenogenic reproduction of the isogenic homozygous transgenic fish. These results demonstrate that a combination of transgenesis and artificial parthenogenesis will make the rapid utilization of genetically pure homozygous transgenic system in vertebrate transgenesis possible.

Evaluation of Japanese quail as a model system for avian transgenesis using avian leukosis viruses

Vertical transmission of avian leukosis viruses (ALV) can occur genetically through the germline for both male and female chickens but only nongenetically or congenitally through the female. We had previously shown that tolerantly ALV-infected males, from ALV injection into fertile chicken eggs at day of set, can transmit proviral DNA to their progeny through the germline. An attempt was made to repeat this successful retroviral germline insertion technique of chickens in Japanese quail. After an initial difficulty of infecting quail chicks in ovo at day of set with high titer nonpathogenic recombinant and pathogenic ALV, adequate numbers of tolerantly ALV-infected quail were produced by injecting ALV-infected chicken embryo fibroblasts (CEF) at day of set. Tolerantly ALV-infected male and female quail were then mated to nonviremic quail and vertical transmission of ALV to progeny chicks was determined by analyzing blood for viral antigens and proviral DNA using standard techniques. Vertical transmission of ALV was only detected in the progeny of viremic females. Thus, little or no germline transmission of ALV to progeny occurred from viremic males. Tolerantly ALV-infected males and females from congenital ALV infection, which should infect the embryo and presumably the primordial germ cells (PGC) earlier than egg injection, were mated to nonviremic quails. Vertical transmission of ALV to progeny chicks was analyzed as before. Again, vertical transmission of ALV was only detected in the progeny of viremic females. We conclude that Japanese quail will not be useful in avian transgenic studies involving ALV retroviral vectors.

Developmental origin of avian primordial germ cells and its unique differentiation in the gonads of mixed-sex chimeras.

Developmental origin of avian primordial germ cells and its unique differentiation in the gonads of mixed-sex chimeras.

A Complete Culture System for Avian Transgenesis, Supporting Quail Embryos from the Single-Cell Stage to Hatching

We report here a method to produce quail hatchlings by culture in vitro from the single-cell stage. The culture is composed of three steps. In the first step, the fertilized ovum surrounded by thick albumen obtained from the magnum is cultured for 24 hr at 41.5°C in a tightly sealed 20-ml plastic cup with chicken thin albumen added to the equator level of the ovum (System Q1). In the second step, a quail egg shell, cut horizontally and emptied, is used as a bed shell. After the thick albumen is removed, the embryo with egg yolk is transferred to the bed shell and thin albumen from chicken eggs is added to fill the shell. Then, the embryo is cultured for an additional 52 hr at 37.5°C while being rocked at an angle of 90 degrees at 30-min intervals (System Q2). The embryo is transferred again to a chicken bed shell and cultured at 37.5°C with rocking at a 30-degree angle (System Q3). Just before hatching, the rocking of embryos is stopped. The procedure yielded a hatchability of 25%. For transgenesis, a plasmid construct containing a β-actin-lacZ hybrid gene (pMiwZ) is microinjected into the ovum at the single-cell stage, which is cultured in vitro for 85-90 hr using Systems Q1 and Q2 consecutively. Seven out of 17 surviving embryos exhibited lacZ gene expression in embryonic tissues as detected by histochemistry. The procedure described here should be highly applicable for the production of transgenic birds.