Transgenic Eggs Research Articles

Application of Recombinant Monoclonal Antibodies from Transgenic Chicken Bioreactors in Enzyme-Linked Immunosorbent Assay.

Transgenic chicken bioreactors can efficiently produce egg whites containing large quantities of recombinant proteins. We previously developed transgenic chickens that produce recombinant monoclonal antibodies (mAbs) against epidermal growth factor receptor 2 (HER2). However, the practical applications of mAbs derived from transgenic eggs have not yet been examined. Therefore, we aimed to evaluate whether these recombinant mAbs can be used in enzyme-linked immunosorbent assay (ELISA). Recombinant HER2 mAbs from transgenic eggs were dissolved in phosphate-buffered saline and applied directly to 96-well microplates as immobilized antibodies without purification. The performance of ELISA using the unpurified recombinant HER2 mAbs from transgenic eggs was comparable to that of ELISA using commercially available purified recombinant HER2 mAbs. Moreover, ELISA using unpurified recombinant HER2 mAbs from transgenic eggs demonstrated high antigen specificity and was successfully applied to samples from cultured cell lysates derived from HER2-positive and HER2-negative cell lines. The unpurified recombinant HER2 mAbs from transgenic eggs were also efficiently used as immobilized antibodies in paper-based ELISA. In conclusion, our findings suggest that recombinant mAbs from transgenic eggs have the potential to be used to develop economic ELISA devices. To the best of our knowledge, this study is the first to use recombinant HER2 mAbs from transgenic eggs in ELISA.

An efficient and safe strategy for germ cell-specific automatic excision of foreign DNA in F1 hybrid transgenic silkworms.

The safety of transgenic technology is a major obstacle in the popularization and use of transgenic silkworms and their products. In sericulture, only the first filial generation (F1 ) hybrid eggs produced by cross-breeding Japanese and Chinese original strains are usually used for the large-scale breeding of silkworms, but this may result in uncontrolled transgene dispersal during the popularization and application of the F1 hybrid transgenic eggs. To address this issue, we developed a safe and efficient strategy using the GAL4/Upstream activating sequence (UAS) system, the FLP/flippase recognition target (FRT) system, and the gonad-specific expression gene promoters (RSHP1p and Nanosp) for the germ cell-specific automatic excision of foreign DNA in the F1 hybrid transgenic silkworms. We established 2 types of activator strains, R1p::GAL4-Gr and Nsp::GAL4-Gr, containing the testis-specific GAL4 gene expression cassettes driven by RSHP1p or Nanosp, respectively, and 1 type of effector strain, UAS::FLP-Rg, containing the UAS-linked FLP gene expression cassette. The FLP recombinase-mediated sperm-specific complete excision of FRT-flanked target DNA in the F1 double-transgenic silkworms resulting from the hybridization of R1p::GAL4-Gr and UAS::FLP-Rg was 100%, whereas the complete excision efficiency resulting from the hybridization of Nsp::GAL4-Gr and UAS::FLP-Rg ranged from 13.73% to 80.3%. Additionally, we identified a gene, sw11114, that is expressed in both testis and ovary of Bombyx mori, and can be used to establish novel gonad-specific expression systems in transgenic silkworms. This strategy has the potential to fundamentally solve the safety issue in the production of F1 transgenic silkworm eggs and provides an important reference for the safety of transgenic technology in other insect species.

Transfer of the zp3a gene results in changes in egg adhesiveness and buoyancy in transgenic zebrafish.

Reproductive strategies and spawning habits play key roles in the evolution of endemic East Asian cyprinids. However, the molecular mechanisms underlying the regulation of spawning habits are not well understood. We recently identified zona pellucida (Zp) as the top differentially expressed protein between East Asian cyprinids that produce adhesive and semi-buoyant eggs, suggesting that Zp protein may play important roles in the regulation of egg type. In this work, we generated transgenic zebrafish in which oocyte-specific expression of zp genes from rare minnow ( Gobiocypris rarus), an East Asian cyprinid laying adhesive eggs, was driven by a zebrafish zp3.2 gene promoter. We found that the transgenic eggs obtained partial adhesiveness and exhibited alteration in hydration and buoyancy. Abnormal metabolism of vitellogenin (VTG) may contribute to enhanced hydration and/or buoyancy. Our work shows that expression of the exogenous zp3a gene from an adhesive-egg producing fish is sufficient to induce changes in both egg adhesiveness and buoyancy in zebrafish, emphasizing the important role of zp genes in the regulation of spawning habits. Our results thus provide new insights into how endemic East Asian cyprinids may have adapted to the Yangtze river-lake system via changes in spawning habits.

Human Zona Pellucida Glycoproteins: Binding Characteristics With Human Spermatozoa and Induction of Acrosome Reaction.

Human zona pellucida (ZP) matrix is composed of four glycoproteins designated as ZP glycoprotein -1 (ZP1), -2 (ZP2), -3 (ZP3), and -4 (ZP4). Mutations in the genes encoding human ZP glycoproteins are one of the causative factors leading to abnormal ZP matrix and infertility in women. Relevance of the human ZP glycoproteins in ‘sperm–oocyte’ binding has been delineated by using either transgenic animal models expressing human zona proteins or purified native/recombinant human zona proteins. Studies based on the purified native/recombinant human zona proteins revealed that ZP1, ZP3, and ZP4 primarily bind to the capacitated acrosome-intact human spermatozoa whereas ZP2 binds to acrosome-reacted spermatozoa. On the contrary, human spermatozoa binds to the eggs obtained from transgenic mouse lines expressing human ZP2 but not to those expressing human ZP1, ZP3, and ZP4 suggesting that ZP2 has an important role in human ‘sperm–oocyte’ binding. Further studies using transgenic mouse lines showed that the N-terminus of human ZP2 mediate the taxon-specific human sperm–oocyte binding. Both glycans and protein-protein interactions have a role in human gamete interaction. Further studies have revealed that the purified native/recombinant human ZP1, ZP3, and ZP4 are competent to induce acrosome reaction. Human sperm binds to the mouse transgenic eggs expressing human ZP1-4 instead of mouse ZP1-3 proteins, penetrated the ZP matrix and accumulated in the perivitelline space, which were acrosome-reacted suggesting that human ZP2 in transgenic mouse model also induce acrosome reaction. In humans N-linked glycosylation of zona proteins have been shown to play an important role in induction of the acrosome reaction. Hence in humans, based on studies using transgenic mouse model as well as purified native/recombinant zona proteins, it is likely that more than one zona protein is involved in the ‘sperm–oocyte’ binding and induction of the acrosome reaction.

Developmental Sensitivity in Schistosoma mansoni to Puromycin To Establish Drug Selection of Transgenic Schistosomes.

Schistosomiasis is considered the most important disease caused by helminth parasites, in terms of morbidity and mortality. Tools to facilitate gain- and loss-of-function approaches can be expected to precipitate the discovery of novel interventions, and drug selection of transgenic schistosomes would facilitate the establishment of stable lines of engineered parasites. Sensitivity of developmental stages of schistosomes to the aminonucleoside antibiotic puromycin was investigated. For the schistosomulum and sporocyst stages, viability was quantified by fluorescence microscopy following dual staining with fluorescein diacetate and propidium iodine. By 6 days in culture, the 50% lethal concentration (LC50) for schistosomula was 19 μg/ml whereas the sporocysts were 45-fold more resilient. Puromycin potently inhibited the development of in vitro-laid eggs (LC50, 68 ng/ml) but was less effective against liver eggs (LC50, 387 μg/ml). Toxicity for adult stages was evaluated using the xCELLigence-based, real-time motility assay (xWORM), which revealed LC50s after 48 h of 4.9 and 17.3 μg/ml for male and female schistosomes, respectively. Also, schistosomula transduced with pseudotyped retrovirus encoding the puromycin resistance marker were partially rescued when cultured in the presence of the antibiotic. Together, these findings will facilitate selection on puromycin of transgenic schistosomes and the enrichment of cultures of transgenic eggs and sporocysts to facilitate the establishment of schistosome transgenic lines. Streamlining schistosome transgenesis with drug selection will open new avenues to understand parasite biology and hopefully lead to new interventions for this neglected tropical disease.

Generation of transgenic chickens expressing the human erythropoietin (hEPO) gene in an oviduct-specific manner: Production of transgenic chicken eggs containing human erythropoietin in egg whites

The transgenic chicken has been considered as a prospective bioreactor for large-scale production of costly pharmaceutical proteins. In the present study, we report successful generation of transgenic hens that lay eggs containing a high concentration of human erythropoietin (hEPO) in the ovalbumin. Using a feline immunodeficiency virus (FIV)-based pseudotyped lentivirus vector enveloped with G glycoproteins of the vesicular stomatitis virus, the replication-defective vector virus carrying the hEPO gene under the control of the chicken ovalbumin promoter was microinjected to the subgerminal cavity of freshly laid chicken eggs (stage X). Stable germline transmission of the hEPO transgene to the G1 progeny, which were non-mosaic and hemizygous for the hEPO gene under the ovalbumin promoter, was confirmed by mating of a G0 rooster with non-transgenic hens. Quantitative analysis of hEPO in the egg whites and in the blood samples taken from G1 transgenic chickens showed 4,810 ~ 6,600 IU/ml (40.1 ~ 55.0 μg/ml) and almost no detectable concentration, respectively, indicating tightly regulated oviduct-specific expression of the hEPO transgene. In terms of biological activity, there was no difference between the recombinant hEPO contained in the transgenic egg white and the commercially available counterpart, in vitro. We suggest that these results imply an important step toward efficient production of human cytokines from a transgenic animal bioreactor.

Purification and Characterization of Recombinant Human Lysozyme from Eggs of Transgenic Chickens.

Transgenic chickens as bioreactors have several advantages, such as the simple establishment procedure, correct glycosylation profile of expressed proteins, etc. Lysozyme is widely used in food industry, livestock farming, and medical field as a replacement of antibiotics because of its antibacterial and complement system-modulating activity. In this study, we used RT-PCR, Western blot, and immunofluorescence to detect the expression of recombinant human lysozyme (rhLY) in the transgenic chicken. We demonstrated that the transgene of rhLY was genetically stable across different generations. We next optimized the purification procedure of rhLY from the transgenic eggs by utilizing two steps of cation-exchange chromatography and one gel-filtration chromatography. About 6 mg rhLY with the purity exceeding 90% was obtained from ten eggs, and the purification efficiency was about 75%. The purified rhLY had similar physicochemical and biological properties in molecular mass and antibacterial activity compared to the commercial human lysozyme. Additionally, both of them exhibited thermal stability at 60°C and tolerated an extensive pH range of 2 to 11. In conclusion, our study proved that the transgenic chickens we have previously generated were genetically stable and suitable for the production of active rhLY. We also provided a pipeline for purifying the recombinant proteins from transgenic eggs, which could be useful for other studies.

Production of germline transgenic chickens expressing enhanced green fluorescent protein using a MoMLV‐ based retrovirus vector

The Moloney murine leukemia virus (MoMLV) -based retrovirus vector system has been used most often in gene transfer work, but has been known to cause silencing of the imported gene in transgenic animals. In the present study, using a MoMLV-based retrovirus vector, we successfully generated a new transgenic chicken line expressing high levels of enhanced green fluorescent protein (eGFP). The level of eGFP expression was conserved after germline transmission and as much as 100 microg of eGFP could be detected per 1 mg of tissue protein. DNA sequencing showed that the transgene had been integrated at chromosome 26 of the G1 and G2 generation transgenic chickens. Owing to the stable integration of the transgene, it is now feasible to produce G3 generation of homozygous eGFP transgenic chickens that will provide 100% transgenic eggs. These results will help establish a useful transgenic chicken model system for studies of embryonic development and for efficient production of transgenic chickens as bioreactors.

Transgenic RNAi-mediated reduction of MSY2 in mouse oocytes results in reduced fertility

MSY2 is implicated in regulating the stability and translation of maternal mRNAs during mouse oogenesis. We report here that by driving the expression of a transgene encoding an Msy2 hairpin dsRNA in growing oocytes using the oocyte-specific Zp3 promoter, the amount of MSY2 protein was reduced by at least 60% in fully grown oocytes. The decrease appeared specific because no decrease was observed in either non-targeted mRNAs or proteins. Fertility of transgenic females was severely reduced. Although transgenic eggs could be inseminated, the eggs did not exhibit the normal series of oscillations in intracellular Ca 2+, resume meiosis, undergo cortical granule exocytosis, or ZP2 cleavage to ZP2 f. Transgenic oocytes also displayed a higher incidence of both the non-surrounded nucleolus chromatin morphology, and abnormal meiotic spindle formation was observed following oocyte maturation. Transgenic oocytes contained less total mRNA (approximately 75–80% that of non-transgenic oocytes) and displayed a reduced level of protein synthesis. Moreover, several of the maturation-associated changes in protein synthesis failed to occur in the transgenic oocytes. These results support a role for MSY2 in stabilizing maternal mRNAs in growing oocytes, a process essential to generate meiotically and developmentally competent oocytes.

Transformation in parasitic nematodes at last?

It is somewhat ironic that nematodes, represented by Caenorhabditis elegans, have been at the forefront of genetic and genomic research for the past 20 years, culminating with the completion of the genomic sequence in 1998. This was the first complete multicellular eukaryote sequence and, of greater general significance perhaps, served as the test-bed for the development of large-scale sequencing technology. However, despite this tremendous basic resource and a rapidly expanding parasitic nematode expressed sequence tag (EST) dataset (http://www.nematode.net), parasitic nematodes have lagged far behind other parasite genera in recent years owing to a lack of molecular tools for functional genomic analysis.The report by Lok and Massey [1xTransgene expression in Strongyloides stercoralis following gonadal microinjection of DNA constructs. Lok, J.B. and Massey, H.C. Mol. Biochem. Parasitol. 2002; 119: 279–284Crossref | PubMed | Scopus (30)See all References[1], the expression of a green fluorescent reporter (GFP) transgene in developing eggs of Strongyloides stercoralis females microinjected with a GFP reporter construct, is a sign of considerable progress. This provides us with several reasons for optimism. First, the use of S. stercoralis is an important development because this parasite provides access to at least one free-living generation and the potential of recovering the first putatively transgenic progeny without the need to infect a host. This removes one of the most challenging barriers to functional genomics in parasitic nematodes of mammals: the near absolute necessity to infect a host to produce the next generation. It is no coincidence that the parasitic protozoa yielded to transgenesis after in vitro culture was made possible. Unfortunately, the apparently transgenic eggs that showed GFP expression during early in utero development failed to hatch, presumably as a result of a lethality associated with the observed high levels of GFP expression. Therefore, if truly heritable transformation is to be achieved (as it is clear from Caenorhabditis elegans that the majority of transformation events following microinjection are not inherited), obtaining a transgenic line or strain will be a numbers game. The high frequency of apparently transgenic eggs observed suggests that the necessary numbers will also be available if the reporter-associated lethality can be overcome, and the ability to culture the injected free-living or heterogonic females in vitro means that it will be possible to recover and screen every viable offspring.Second, the authors used endogenous promoters from highly expressed S. stercoralis genes to drive reporter gene expression. The promoter sequences were isolated using complementary DNA (cDNA) sequence as a starting point. The availability of many thousands of parasite ESTs suggests that obtaining other promoters will not be a limiting factor in the development of the technologyFinally, the promising results reported here will, hopefully, encourage other labs (perhaps using other species with in vitro culture potential) and the relevant funding agencies to take up the challenge of developing a system for generating truly heritable transgenesis. It is clearly a feasible goal and one that is worth pursuing.

The tyrosinase gene from medakafish: transgenic expression rescues albino mutation.

We have determined the 9.8 kb genomic nucleotide sequence of the tyrosinase gene and its 5 upstream region from a teleost, medakafish (Oryzias latipes), and shown that the coding region is composed of five exons and four introns, spanning 4.7 kb. While the number and sizes of the exons were found to be similar to those of mammalian tyrosinase genes, however, the total size of the coding region (4.7 kb) was demonstrated to be less than one tenth those of mouse (ca. 70 kb) and human (> 70 kb) genes. Primer extension analysis revealed that the transcription initiation site starts with a long untranslated leader sequence (340 nucleotide long) from the AUG start codon. A characteristic CATGTG sequence known as a putative regulatory motif in melanocyte-specific genes was present in the 131st base upstream from the initiation site, while other typical regulatory elements such as the TATA-box or M-box common to terrestrial vertebrates were lacking. Transgenic experiments were carried out by microinjecting two kinds of plasmid clones into fertilized eggs of the albino i(l) mutant: one consisting of the genomic tyrosinase gene with the 10 kb 5 upstream region and the other the tyrosinase cDNA with the 3 kb 5 upstream region. The results showed that 53 and 45 of 114 and 118 transgenic eggs, respectively, developed normally beyond hatching and 15 and 10 exhibited a mosaic pattern of pigmentation. Despite the absence of typical regulatory elements like a TATA-box in both cases correct melanin pigmentation was obtained without ectopic expression. Thus, transgenic expression rescued from the albino-i(l) mutation, and the i locus of the medaka genome can be concluded to encode the tyrosinase gene.