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Despite great values in many applications, heavy chain-only antibodies (HcAbs) are naturally only produced in camelids and sharks, which are not easy to access and handle. Production of the type of antibodies in small laboratory animals would remarkably facilitate their applications. We previously reported a mouse line in which the CH1 exon of mouse γ1 was deleted that could express heavy chain-only IgG1 antibodies. However, these mice showed an extremely weak IgG1 response to specific antigens when immunized, and we could only achieve single VH domains with low affinity to antigens using these mice. One possibility is that the mouse germline VH repertoire was not sufficient to support the expression of functional heavy chain-only antibodies. In this study, we report the generation of a rat line in which the CH1 exon of the γ2a gene was removed and the γ1 and γ2b genes were silenced. Although the genetically modified rats expressed heavy chain-only IgG2a, they also exhibited a very weak IgG2a response to antigen immunization. Panning of a phage library constructed using IgG2a VH segments amplified from immunized rats identified antigen-specific single VH antibodies, which also exhibited much lower affinity than that of commercial mAbs. Together with our previous report, this study suggests that the simple genetic removal of the CH1 exon does not guarantee the successful expression of functional heavy chain-only antibodies. [ABSTRACT FROM AUTHOR]

Long Evans rat strains are applied as research models in a broad spectrum of biomedical fields (>15,800 citations, NCBI PubMed). Here, we report an approach to genetically modify the Long Evans rat germline in donor spermatogonial stem cells. Long Evans rat spermatogonial lines were derived from freshly isolated laminin-binding spermatogonia. Laminin-binding spermatogonia were cultured over multiple passages on fibroblast feeder layers in serum-free culture medium containing GDNF and FGF2. Long Evans rat spermatogonial lines were genetically modified by transposon transduction to express a germline, tdTomato reporter gene. Donor rat spermatogonial lines robustly regenerated spermatogenesis after transplantation into testes of busulfan-treated, allogenic, Long Evans rats. Donor-derived spermatogenesis largely restored testis size in the chemically sterilized, recipient Long Evans rats. Recipient Long Evans rats stably transmitted the tdTomato germline marker to subsequent generations. Overall, Long Evans rat spermatogonial lines provided effective donor germline vectors for genetically modifying Long Evans rats. [ABSTRACT FROM AUTHOR]

doptive cell transfer studies in regenerative research and identification of genetically modified cells after gene therapy in vivo require unequivocally identifying and tracking the donor cells in the host tissues, ideally over several days or for up to several months. The use of reporter genes allows identifying the transferred cells but unfortunately most are immunogenic to wild-type hosts and thus trigger rejection in few days. The availability of transgenic animals from the same strain that would express either high levels of the transgene to identify the cells or low levels but that would be tolerant to the transgene would allow performing long-term analysis of labelled cells. Herein, using lentiviral vectors we develop two new lines of GFP-expressing transgenic rats displaying different levels and patterns of GFP-expression. The “high-expresser” line (GFP) displayed high expression in most tissues, including adult neurons and neural precursors, mesenchymal stem cells and in all leukocytes subtypes analysed, including myeloid and plasmacytoid dendritic cells, cells that have not or only poorly characterized in previous GFP-transgenic rats. These GFP-transgenic rats could be useful for transplantation and immunological studies using GFP-positive cells/tissue. The “low-expresser” line expressed very low levels of GFP only in the liver and in less than 5% of lymphoid cells. We demonstrate these animals did not develop detectable humoral and cellular immune responses against both transferred GFP-positive splenocytes and lentivirus-mediated GFP gene transfer. Thus, these GFP-transgenic rats represent useful tools for regenerative medicine and gene therapy. [ABSTRACT FROM AUTHOR]

The objective of the present study was to produce rat offspring by intracytoplasmic sperm injection (ICSI) using a Piezo-driven micromanipulator. Transgenic male rats carrying a green fluorescent protein gene (GFP: homozygous) were used as sperm donors. The epididymal spermatozoa were suspended and sonicated in m-KRB medium and were frozen in the same medium at −20°C until use. When the sperm heads were aspirated into injection pipettes 7–10 μm in diameter and introduced into oocytes from the Wistar strain, no offspring resulted from the transfer of 59 eggs. In contrast, the sperm heads were hung on the tip of injection pipettes 2–4 μm in diameter and introduced into the oocytes, use of Piezo resulting in the production of 18 transgenic offspring carrying the GFP gene from 181 eggs transferred. The oocytes from the Sprague–Dawley strain also supported full-term development following ICSI with three offspring resulting from 163 transferred eggs. In an additional ICSI trial, spermatozoa from infertile transgenic rats carrying human lactalbumin with the thymidine kinase gene (LAC3: heterozygous) were used. The spermatozoa of the LAC3 transgenic rats appeared to be defective and immotile because of the expression of thymidine kinase in the testes, and no ICSI offspring resulted from 218 transferred eggs. These results suggest that ICSI is applicable in rats when Piezo-driven smaller pipettes are used to inject sperm heads together with a limited amount of the surrounding medium and that the ability of isolated sperm heads to participate in normal embryo development is maintained under the cryopreservation conditions employed. [ABSTRACT FROM AUTHOR]

Biopharmaceuticals intended for humans are immunogenic in animals. Antibodies associated with their administration make it difficult to perform repeated-dose pharmacology and toxicology studies in animals. Despite suggestions to solve this problem with transgenic animal technology, an effective strategy has not yet been reported. The objective of the present study was to provide an efficient strategy to develop rats tolerant to biopharmaceuticals such as human gene-based proteins. The present study used transgenic rat lines (lines 311-6, 308-5, and 305-1) carrying a fusion gene designed to express the human growth hormone (hGH) gene under the control of the bovine αS1 casein gene promoter. Three lactating females with the transgene, produced approximately 4 mg/ml, 300 μg/ml, and 10 ng/ml in their milk. Male 8-week-old rats from these three lines were immunized with hGH three times (week 0, 1, and 3 ) and the production of antibodies against hGH in their sera were examined at week 4. While the hGH serum antibody titers increased over 1000-fold in wild-type control rats, there was no detectable antibody against hGH in the sera of these three transgenic lines. Human growth hormone in their sera was undetectable (lines 308-5 and 305-1) or much lower than the endogenous biologic level of rat growth hormone (line 311-6). Importantly, lines 308-5 and 305-1 developed tolerance to hGH without detectable hGH in their sera and these lines will be very useful for the repeated dose pharmacology and toxicology studies. These results suggest that a milk protein promoter can be a useful tool to develop transgenic rats that are tolerant to biopharmaceuticals intended for humans. [ABSTRACT FROM AUTHOR]

We investigated the application of cryopreserved pronuclear stage zygotes for the production of transgenic rats. Most of the pronuclear stage zygotes cryopreserved by conventional two step freezing or vitrification appeared morphologically normal, but the proportion of frozen zygotes that developed into fetuses following transfer (59.7–60.2%) was higher than that of vitrified zygotes (5.5–22.1%). When the frozen thawed zygotes were used for DNA microinjection, 97.5% survived after DNA microinjection and 25.1% of the transferred zygotes developed into fetuses. These proportions were comparable to those of the fresh control zygotes (97.0% and 30.0%, respectively). The integration efficiency of the exogenous DNA into fetuses was similar between the frozen group (3.3% per injected zygote) and the control group (3.5%). These results indicate that pronuclear stage rat zygotes can be successfully cryopreserved by conventional two step freezing for production of transgenic rats. [ABSTRACT FROM AUTHOR]

The investigation of human complement (C) inhibitors with a view to overcoming C-mediated tissue injury stands to benefit from the production of anatomically suitable transgenic animals. In this study, we used the CMV-IE1 enhancer/promoter to control the expression in vivo in transgenic rats of the human terminal C protein inhibitor CD59. Five transgenic rats were identified, of which four possessed at least one complete copy of the transgene. The presence of human CD59 transcripts and protein was demonstrated in two transgenic rat lines. A widespread tissue distribution of cells expressing human CD59, similar in the two lines, was observed-principally in pancreas, brain, heart, kidney, intestine and striated muscle. Whereas expression in pancreas and brain was uniform, mosaicism of CD59 expression was observed in some tissues such as heart and kidney, a proportion of cells within the tissue not expressing the transgene. Immunohistological analysis revealed surface expression of human CD59 in a variety of cells, including fibroblasts, epithelial cells and muscle cells, but not in endothelial cells. In conclusion, this paper analyses at the cellular level human CD59 expression directed by the CMV promoter in transgenic rats, and discusses how they could be used to investigate in vivo the role of C in a variety of pathologies. [ABSTRACT FROM AUTHOR]