Zsuzsanna Bösze

Expression of the neonatal Fc receptor (FcRn) in the bovine mammary gland

In ruminants, protective immunoglobulins are transferred to the newborn via colostrum to mediate maternal immunity. There is a high selectivity in the transport of immunoglobulins from the maternal plasma across the mammary barrier into the colostrum, and only IgG1 is transferred in large amounts. We have recently analysed the expression of the neonatal Fc receptor (FcRn) in sheep mammary gland around parturition. Re-analysing this issue in bovine confirmed our previous data indicating that FcRn is homogeneously localized in the mammary gland acinar cells before parturition, however a remarkable difference was observed in the pattern after calving, where only the apical side of the cells was strongly stained. The presence of the FcRn in the acinar epithelial cells of the mammary gland and the obvious change in distribution before and after parturition indicate that FcRn plays an important role in the IgG transport during colostrum formation in ruminants.

The transgenic rabbit as model for human diseases and as a source of biologically active recombinant proteins

Until recently, transgenic rabbits were produced exclusively by pronuclear microinjection which results in additive random insertional transgenesis; however, progress in somatic cell cloning based on nuclear transfer will soon make it possible to produce rabbits with modifications to specific genes by the combination of homologous recombination and subsequent prescreening of nuclear donor cells. Transgenic rabbits have been found to be excellent animal models for inherited and acquired human diseases including hypertrophic cardiomyopathy, perturbed lipoprotein metabolism and atherosclerosis. Transgenic rabbits have also proved to be suitable bioreactors for the production of recombinant protein both on an experimental and a commercial scale. This review summarizes recent research based on the transgenic rabbit model.

Germline transgenesis in rabbits by pronuclear microinjection of Sleeping Beauty transposons

The laboratory rabbit (Oryctolagus cuniculus) is widely used as a model for a variety of inherited and acquired human diseases. In addition, the rabbit is the smallest livestock animal that is used to transgenically produce pharmaceutical proteins in its milk. Here we describe a protocol for high-efficiency germline transgenesis and sustained transgene expression in rabbits by using the Sleeping Beauty (SB) transposon system. The protocol is based on co-injection into the pronuclei of fertilized oocytes of synthetic mRNA encoding the SB100X hyperactive transposase together with plasmid DNA carrying a transgene construct flanked by binding sites for the transposase. The translation of the transposase mRNA is followed by enzyme-mediated excision of the transgene cassette from the plasmids and its permanent genomic insertion to produce stable transgenic animals. Generation of a germline-transgenic founder animal by using this protocol takes ∼2 months. Transposon-mediated transgenesis compares favorably in terms of both efficiency and reliable transgene expression with classic pronuclear microinjection, and it offers comparable efficacies (numbers of transgenic founders obtained per injected embryo) to lentiviral approaches, without limitations on vector design, issues of transgene silencing, and the toxicity and biosafety concerns of working with viral vectors.

Neonatal FcR Overexpression Boosts Humoral Immune Response in Transgenic Mice

The neonatal FcR (FcRn) regulates IgG and albumin homeostasis, mediates maternal IgG transport, takes active part in phagocytosis, and delivers Ag for presentation. We have previously shown that overexpression of FcRn in transgenic (Tg) mice extends the half-life of mouse IgG by reducing its clearance. In this paper, we demonstrate that immunization of these mice with OVA and trinitrophenyl-conjugated human IgG results in a 3- to 10-fold increase of Ag-specific IgM and IgG in serum. The IgM increase was unexpected because FcRn does not bind IgM. Our results showed that the affinity of the Ag-specific IgG was at least as good in Tg mice as in the wild-type (wt) controls, implying appropriate affinity maturation in both groups. Influenza vaccination produced a 2-fold increase in the amount of virus-specific Ab in Tg animals, which proved twice as efficient in a hemagglutination inhibition assay as was the case in wt controls. After immunization, Tg mice displayed significantly larger spleens containing a higher number of Ag-specific B cells and plasma cells, as well as many more granulocytes and dendritic cells, analyzed by ELISPOT and flow cytometric studies. The neutrophils from these Tg mice expressed the Tg FcRn and phagocytosed IgG immune complexes more efficiently than did those from wt mice. These results show that FcRn overexpression not only extends the IgG half-life but also enhances the expansion of Ag-specific B cells and plasma cells. Although both effects increase the level of Ag-specific IgG, the increase in immune response and IgG production seems to be more prominent compared with the reduced IgG clearance.

Induced pluripotent stem cells derived from rabbits exhibit some characteristics of naïve pluripotency

Summary Not much is known about the molecular and functional features of pluripotent stem cells (PSCs) in rabbits. To address this, we derived and characterized 2 types of rabbit PSCs from the same breed of New Zealand White rabbits: 4 lines of embryonic stem cells (rbESCs), and 3 lines of induced PSCs (rbiPSCs) that were obtained by reprogramming adult skin fibroblasts. All cell lines required fibroblast growth factor 2 for their growth and proliferation. All rbESC lines showed molecular and functional properties typically associated with primed pluripotency. The cell cycle of rbESCs had a prolonged G1 phase and a DNA damage checkpoint before entry into the S phase, which are the 2 features typically associated with the somatic cell cycle. In contrast, the rbiPSC lines exhibited some characteristics of naïve pluripotency, including resistance to single-cell dissociation by trypsin, robust activity of the distal enhancer of the mouse Oct4 gene, and expression of naïve pluripotency-specific genes, as defined in rodents. According to gene expression profiles, rbiPSCs were closer to the rabbit inner cell mass (ICM) than rbESCs. Furthermore, rbiPSCs were capable of colonizing the ICM after aggregation with morulas. Therefore, we propose that rbiPSCs self-renew in an intermediate state between naïve and primed pluripotency, which represents a key step toward the generation of bona fide naïve PSC lines in rabbits.

Producing recombinant human milk proteins in the milk of livestock species

Recombinant human proteins produced by the mammary glands of genetically modified transgenic livestock mammals represent a special aspect of milk bioactive components. For therapeutic applications, the often complex posttranslational modifications of human proteins should be recapitulated in the recombinant products. Compared to alternative production methods, mammary gland production is a viable option, underlined by a number of transgenic livestock animal models producing abundant biologically active foreign proteins in their milk. Recombinant proteins isolated from milk have reached different phases of clinical trials, with the first marketing approval for human therapeutic applications from the EMEA achieved in 2006.

Effect of rabbit κ-casein expression on the properties of milk from transgenic mice

Transgenic mice were produced carrying the coding region of the rabbit kappa-casein gene linked to the upstream region of the rabbit whey acidic protein gene. Mice from the highest-expressing line produced 2.5 mg rabbit kappa-casein/ml in their milk. The foreign protein was associated with the casein micelles and altered micelle size, though in the high-expressing line rabbit kappa-casein also segregated into the whey fraction obtained after centrifuging the milk samples. Milk from transgenic mice had the same overall protein content as that from non-transgenic mice, except for the transgene product. However, litters fed with this transgenic mouse milk grew less well than litters given milk from non-transgenic mice. This reduction in growth was not related to changes in mammary gland structure or mammary cell morphology. Preliminary results indicated that milk from the transgenic mice had a higher viscosity.

Recombinant protein expression in milk of livestock species.

Producing complex recombinant proteins in the milk of transgenic animals offers several advantages: large amounts of proteins can be obtained, and in most cases, these proteins are properly folded, assembled, cleaved, and glycosylated. The level of expression of foreign genes in the mammalian gland cannot be predicted in all cases, and appropriate vectors must be used. The main elements of these vectors are as follows: a well-characterized specific promoter, the coding region of the gene of interest, preferably with a homologous or heterologous intron, to improve transcription efficiency, and an insulator or boundary element to counteract the chromosomal position effects at the integration site. Once high expression levels are achieved, and the recombinant protein is purified, an essential step in the analysis of the final product is determining its degree of glycosylation. This is an important readout because it can affect among other parameters the stability and immunogenicity of the recombinant protein.

Polymorphism of the rabbit kappa kasein gene and its influence on performance traits

The rabbit κ-casein encoding gene has previously been shown to possess two alleles. The two alleles do not differ in their coding region and in the accumulation levels of mRNA. However they differ greatly with respect to their intronic regions. The rearranged regions in the first and fourth introns were found to be inverse and complementary LINE sequences. The A allele was found to be more frequent in different European breeds. Correlation of the κ-casein genotype with the breeding capacity in a New Zealand White rabbit stock has been examined.

Placenta-specific gene manipulation in rabbits

Lentiviral gene constructs can be efficiently and specifically delivered to trophoblast cell lineages in rodents. In vivo genetic manipulation of trophoblast cell lines enables functional and developmental studies in the placenta. In this report we show that genetic modification can be produced in the extraembryonic tissues of rabbits by lentiviral gene constructs. When 8-16 cell stage embryos were injected with lentiviral particles, strong reporter gene expression resulted in the rabbit placenta. The expression pattern displayed some mosaicism. A strikingly high degree of mosaic GFP expression was detected in some parts of the yolk sac, which is a hypoblast-derived tissue. Whereas expression of the reporter gene construct was detected in placentas and yolk sacs, fetuses never expressed the transgene. As rabbits are an ideal model for functional studies in the placenta, our method would open new possibilities in rabbit biotechnology and placentation studies.