Herman A. de Boer

Expression of human lactoferrin in milk of transgenic mice

The expression of human lactoferrin (hLF) in the milk of transgenic mice is described. Regulatory sequences derived from the bovine αS1-casein gene were fused to the coding sequence of the hLF cDNA and several lines of transgenic mice were generated. Human LF RNA was detected exclusively in the mammary gland of lactating females and only after the onset of lactation. No aberrant RNA products could be detected using northern blotting and primer extension analysis. The hLF concentrations in the milk ranged from less than 0.1 to 36 μg ml−1. Human LF thus expressed did not differ from human milk derived LF, with respect to molecular mass and immunoreactivity with monoclonal and polyclonal antibodies.

Characterization of Recombinant Human Lactoferrin Secreted in Milk of Transgenic Mice

Human lactoferrin (hLF) is an iron-binding protein involved in host defense against infection and severe inflammation. Transgenic mice were produced harboring either hLF cDNA or genomic hLF sequences fused to regulatory elements of the bovine αS1 casein gene. Recombinant hLF expressed in the milk of transgenic mice (transgenic hLF) was compared with natural (human milk-derived) hLF. Immunological identity of the two forms was shown by double antibody immunoassays and the absence of an anti-hLF antibody response in transgenic mice on hyperimmunization with natural hLF. Mono S cation-exchange chromatography and N-terminal protein sequencing of transgenic and natural hLF revealed identical cationicity and N-terminal sequences. SDS-polyacrylamide gel electrophoresis and absorbance measurements of purified transgenic hLF showed this protein was 90% saturated with iron, whereas natural hLF is only 3% saturated. The pH-mediated release of iron from transgenic hLF was not different from that of iron-saturated natural hLF. Unsaturated transgenic hLF could be completely resaturated upon addition of iron. Slight differences in mobility between transgenic and natural hLF on SDS-polyacrylamide gel electrophoresis were abolished by enzymatic deglycosylation. Binding of transgenic and natural hLF to a range of ligands, including bacterial lipopolysaccharide, heparin, single-stranded DNA, Cibacron blue FG 3A, and lectins, was not different. Based on these observations, we anticipate that (unsaturated) rhLF and natural hLF will exert similar, if not identical, antibacterial and anti-inflammatory activity in vivo.

Expression of cDNA-encoded human acid α-glucosidase in milk of transgenic mice

Enzyme replacement therapy is at present the option of choice for treatment of lysosomal storage diseases. To explore the feasibility of lysosomal enzyme production in milk of transgenic animals, the human acid α-glucosidase cDNA was placed under control of the αS1-casein promoter and expressed in mice. The milk contained recombinant enzyme at a concentration up to 1.5 μg/ml. Enzyme purified from milk of transgenic mice was internalized via the mannose 6-phosphate receptor and corrected enzyme deficiency in fibroblasts from patients. We conclude that transgenically produced human acid α-glucosidase meets the criteria for therapeutic application.

Cloning and characterization of the bovine polymeric immunoglobulin receptor-encoding cDNA

Trans-epithelial transport of polymeric immunoglobulins (pIg) into mucosal and glandular secretions is carried out by the pIg receptor (pIgR). Therefore, expression of the pIgR gene in epithelial cells of mucosal and glandular tissues is an absolute requirement for achieving mucosal immunity. We report the cloning and characterization of the bovine pIgR cDNA. Three overlapping cDNA clones with a total length of 3608 bp yielded an open reading frame encoding a 757-amino-acid (aa) transmembrane (TM) glycoprotein. Although polymorphism was found in two separate clones, Northern blot analysis showed a single pIgR mRNA (approx. 3.8 kb) to be present in the mammary gland, liver, lung, kidney and intestine of a lactating cow. There was no detectable expression of pIgR in the spleen of the same animal. Comparison of the deduced bovine pIgR as sequence with those of rat, mouse, man and rabbit shows that this receptor is highly conserved both in aa sequence and structural organization. The degree of conservation in the TM sequence and the C-terminal cytoplasmic tail, which contains the various signals for intracellular trafficking of the receptor, is 65-73%. We also find a high degree of conservation (61-66%) in the ectoplasmic part of the receptor, known as the secretory component (SC), with an exception for that of the rabbit SC, which is much lower (47%). Among the five Ig-like domains in the SC, the N-terminal domain I, where the primary pIg-binding site is located, showed the highest (72-83%) aa sequence conservation.

High-level expression of bovine αs1-casein in milk of transgenic mice

The bovine αs1-casein gene, isolated from a cosmid library, was introduced into the murine germline. Transgene expression occurred in all transgenic mice, and was confined to the lactating mammary gland. Half of the mouse lines (five out of ten) expressed at relatively high expression levels (>1 mg ml−1). The highest levels of expression were obtained with a transgene containing 14.2 kb of 5′ flanking sequence, in two cases expression levels comparable to (10 mg ml−1) or well above (20 mg ml−1) αs1-casein levels in bovine milk were obtained. Transcription initiation occurred at the same site in the bovine αs1-casein gene in transgenic mouse as in the cow. A marked induction of expression occurred at parturition rather than at mid-pregnancy, and thus resembled the bovine rather than the murine developmental expression pattern. Bovine αs1-casein specific immunoblotting and RIA were developed for characterization and quantificatio n of the recombinant protein. Using these assays, the properties of the recombinant protein could not be distinguished from those of the natural bovine protein. In spite of the high-level tissue-specific and correctly regulated developmental expression of the transgene, expression levels were integration-site dependent. This may indicate that not all cis-acting regulatory elements involved in bovine αs1-casein expression were included in the transgene

Expression of a functional mouse-human chimeric anti-CD19 antibody in the milk of transgenic mice.

Human B cell lymphomas are suitable targets for immunotherapy. Clinical trials with mouse-human chimeric B cell-specific monoclonal antibodies (mAbs) have already shown promising results. However, limitations for their use in clinical trials can be the lack of sufficient amounts and high production costs. Expression of mAbs in the mammary gland of transgenic animals provides an economically advantageous possibility for production of sufficient quantities of a promising antibody for clinical trials and beyond. In this paper, we show the feasibility of this approach, by generating transgenic mice expressing mouse-human chimeric anti-CD19 mAbs in their milk. Mouse anti-CD19 variable (V) region genes were combined with human IgG1 heavy (H) and kappa light (L) chain constant (C) region genes and fused to the bovine β-lactoglobulin (BLG) promoter in two separate expression cassettes. Co-injection resulted in five transgenic lines. In one of these lines completely assembled chimeric mAbs were secreted into the milk, at an approximate level of 0.5mg/ml. These mAbs were able to bind specifically to the CD19 surface antigen on human B cells.

Mammary gland-specific hypomethylation ofHpa II sites flanking the bovine αS1-casein gene

In the lactating cow, mammary gland-specific hypomethylation occurs at twoHpa II sites in the 5′-flanking region of the αS1-casein gene, and one in the 3′-region. These sites, A, B. and C, are at nucleotide position −1388, −774 and +18034, respectively, relative to the major transcription start site. Site B was hypomethylated when the αS1-casein gene was expressed, and methylated when not expressed. In transgenic mice containing the bovine αS1-casein 5′ and 3′ regulatory elements fused to the human lactoferrin (hLF) cDNA, in some cases similar methylation patterns of sites A and B, as compared to the situation in the cow, were observed. In five mouse lines (out of the seven analysed) expressing the transgene in the milk, site B was hypomethylated in the mammary gland, while it was methylated in liver. In the two other mouse lines, no correlation was found between transgene expression and mammary gland-specific hypomethylation of site B. One of the five mouse lines with transgene expression and showing mammary-gland-specific hypomethylation of site B was studied in detail. In this mouse line, induction of transgene expression preceded hypomethylation of site B.

Breakdown of ppGpp in spoT+ and spoT− cells of Escherichia coli Manganese and energy requirement and tetracycline inhibition

The reaction pathway and the mechanism of degradation of the regulating nucleotide guanosine 3’.diphosphate, 5’.diphosphate (PpGpp), are unknown. The intracellular ppGpp concentration is controlled by regulation of the rate of its synthesis [l-3] and the rate of its breakdown [4-71. Elucidation of the mechanism of ppGpp synthesis [2,8] has led to the understanding of its control mechanism. Obviously, the same will hold for ppGpp breakdown. In vivo, ppGpp breakdown is a rapid process. Firstorder degradation rate constants up to 3.0 min-’ have been found [l-l 51. However, ppGpp is not broken down in permeabilised cells [ 161, a preparation of membrane vesicles [ 171, in vitro systems for synthesis of ribosomal RNA [ 181, or ppGpp or proteins and in crude cell extracts (unpublished observations). Only in the cold-shocked cell preparations of RaucZ and Cashel [ 111, previously synthesized intracellular ppGpp is broken down. Unfortunately, added ppGpp did not penetrate the remaining cell-wall barrier [ 1 I] . Obviously, the in vitro systems lack necessary components which have been lost or inactivated during preparation. Their nature may become clearer by studying ppGpp breakdown in vivo. As has been shown several times [4-71 the rate of ppGpp breakdown is related to the rate of highenergy phosphate bond production. Mutants (SPOT) which are impaired in ppGpp breakdown (kdepr = 0.1 min-’ ) have been described [9-15,191. Hence, the SPOT gene product is probably an enzyme or a component of an enzyme [20], catalysing ppGpp breakdown [9-15,191.

Over-expression of the murine polymeric immunoglobulin receptor gene in the mammary gland of transgenic mice

The polymeric immunoglobulin receptor (pIgR), a transmembrane protein, transports dimeric IgA (dIgA) across the epithelial cells of the mucosal surfaces into the external secretions, for example milk from the mammary glands. The pIgR is consumed during the transcytosis of dIgA and is cleaved at the apical side of the epithelial cells, regardless of the binding to its ligand (dIgA), to form secretory component (SC). We hypothesize that the expression level of the endogenous murine pIgR gene in the epithelial cells is rate‐limiting for the transport of dIgA across the epithelial cells into the secretions. We address this key issue by generating transgenic mice over‐expressing the pIgR gene in their mammary glands in order to examine the effect on dIgA levels in the milk. Here we report on the generation of transgenic mice and analysis of the expression level of pIgR in their mammary glands. We cloned and characterized the murine pIgR gene and constructed an expression cassette bearing the pIgR gene under the control of the regulatory sequences of the bovine αs1‐casein gene. Four transgenic lines were made, expressing the pIgR construct at RNA and protein level only in their mammary glands. The levels of the SC protein in the milk ranged from 0.1 to 2.7 mg/ml during mid‐lactation. These levels are 10–270 times higher than wild‐type SC levels (0.01 mg/ml).

Ribosomal and non-ribosomal RNA synthesis in vitro

The synthesis of total and ribosomal RNA using nucleoids of Escherichia coli as template was measured; of the total RNA synthesized by endogenous RNA polymerase which only completes chains, and added RNA polymerase which initiates new chains, 50-70 and 3-5%, repectively, was rRNA. Total RNA synthesis by added enzyme, however, was 10-20 times higher than endogenous RNA synthesis; thus rRNA was synthesized at the same rate by the endogenous and the added enzyme. We conclude that the percentage rRNA in vitro is no measure of the rate of rRNA synthesis. Furthermore, it follows that the added enzyme, like the endogenous one, is packed at the physical limit on the ribosomal cistrons. Consequently, initiation of ribosomal cistrons by added enzyme was at or near the maximal rate possible for this system in which the elongation rate is 10-20% of that in vitro. When RNA synthesis was assayed at various ratios of RNA polymerase to phenol-extracted DNA, the amount of rRNA made per DNA, which is a measure of the frequency of transcription of ribosomal cistrons, varied. The ratio of rRNA synthesis relative to total RNA synthesis also varied, but in a different way, again leading to the conclusion that this ratio, as determined in vitro, does not reflect the efficiency of transcription of the ribosomal cistrons.