Frank R. Pieper

Homologous recombination in mammalian cells

The invention relates to methods for intracellularly producing DNA segments by homologous recombination of smaller overlapping DNA fragments and transgenic mammalian cells and transgenic non-human mammals produced by such methods.

Large scale production of recombinant human lactoferrin in the milk of transgenic cows

The limited capacity of current bioreactors has led the biopharmaceutical industry to investigate alternative protein expression systems. The milk of transgenic cattle may provide an attractive vehicle for large-scale production of biopharmaceuticals, but there have been no reports on the characteristics of such recombinant proteins. Here we describe the production of recombinant human lactoferrin (rhLF), an iron-binding glycoprotein involved in innate host defense, at gram per liter concentrations in bovine milk. Natural hLF from human milk and rhLF had identical iron-binding and -release properties. Although natural hLF and rhLF underwent differential N-linked glycosylation, they were equally effective in three different in vivo infection models employing immunocompetent and leukocytopenic mice, and showed similar localization at sites of infection. Taken together, the results illustrate the potential of transgenic cattle in the large-scale production of biopharmaceuticals.

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.

Generation of transgenic mice and germline transmission of a mammalian artificial chromosome introduced into embryos by pronuclear microinjection.

We have generated transgenic mice by pronuclear microinjection of a murine satellite DNA-based artificial chromosome (SATAC). As 50% of the founder progeny were SATAC-positive, this demonstrates that SATAC transmission through the germline had occurred. FISH analyses of metaphase chromosomes from mitogen-activated peripheral blood lymphocytes from both the founder and progeny revealed that the SATAC was maintained as a discrete chromosome and that it had not integrated into an endogenous chromosome. To our knowledge, this is the first report of the germline transmission of a genetically engineered mammalian artificial chromosome within transgenic animals generated through pronuclear microinjection. We have also shown that murine SATACs can be similarly introduced into bovine embryos. The use of embryo microinjection to generate transgenic mammals carrying genetically engineered chromosomes provides a novel method by which the unique advantages of chromosome-based gene delivery systems can be exploited.

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.

Production of recombinant human type I procollagen homotrimer in the mammary gland of transgenic mice.

The large scale production of recombinant collagen for use in biomaterials requires an efficient expression system capable of processing a large (>400 Kd) multisubunit protein requiring post-translational modifications. To investigate whether the mammary gland of transgenic animals fulfills these requirements, transgenic mice were generated containing the αS1-casein mammary gland-specific promoter operatively linked to 37 Kb of the human α1(I) procollagen structural gene and 3′ flanking region. The frequency of transgenic lines established was 12%. High levels of soluble triple helical homotrimeric [(α1)3] type I procollagen were detected (up to 8 mg/ml) exclusively in the milk of six out of 9 lines of lactating transgenic mice. The transgene-derived human procollagen chains underwent efficient assembly into a triple helical structure. Although proline or lysine hydroxylation has never been described for any milk protein, procollagen was detected with these post-translational modifications. The procollagen was stable in mil; minimal degradation was observed. These results show that the mammary gland is capable of expressing a large procollagen gene construct, efficiently assembling the individual polypeptide chains into a stable triple helix, and secreting the intact molecule into the milk.

Intermediate filaments: known structure, unknown function

Intermediate filaments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245 A. IF structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245 B. IF assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 C. Post-translational control of IF assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Association of mRNA and eIF-2α with the cytoskeleton in cells lacking vimentin

The human bladder carcinoma cell lines RT4 and T24 and the human breast adenocarcinoma cell line MCF-7 were found to be negative for vimentin when studied by means of immunofluorescence and immunoblotting. Northern blot analysis revealed that these cells lacked detectable levels of vimentin mRNA with the exception of T24, which contains trace amounts of vimentin mRNA compared to the RNA level in vimentin-containing HeLa cells. CAT assays performed on these cells showed that a hamster vimentin promoter is inactive in RT4 and MCF-7 cells. In the vimentin-lacking cells, the binding of polyribosomes, specific mRNAs, and translation factor eIF-2 alpha to the cytoskeletal fraction was examined. Our results indicate that the presence of a vimentin network is not crucial for the association of the translation machinery with the cytoskeleton. Furthermore, in these vimentin-negative cell lines the immunofluorescence staining pattern of eIF-2 alpha shows a fibro-granular structure that has no resemblance to the cytokeratin or actin cytoskeleton present in these cells.

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.

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