A virus-neutralising monoclonal antibody expressed in the milk of transgenic mice.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
C B Whitelaw
Publications and source records attributed to C B Whitelaw.
Virus-neutralizing monoclonal antibody expressed in milk of transgenic mice provides full protection against virus-induced encephalitis.
Neutralizing antibodies represent a major host defense mechanism against viral infections. In mammals, passive immunity is provided by neutralizing antibodies passed to the offspring via the placenta or the milk as immunoglobulin G and secreted immunoglobulin A. With the long-term goal of producing virus-resistant livestock, we have generated mice carrying transgenes that encode the light and heavy chains of an antibody that is able to neutralize the neurotropic JHM strain of murine hepatitis virus (MHV-JHM). MHV-JHM causes acute encephalitis and acute and chronic demyelination in susceptible strains of mice and rats. Transgene expression was targeted to the lactating mammary gland by using the ovine beta-lactoglobulin promoter. Milk from these transgenic mice contained up to 0.7 mg of recombinant antibody/ml. In vitro analysis of milk derived from different transgenic lines revealed a linear correlation between antibody expression and virus-neutralizing activity, indicating that the recombinant antibody is the major determinant of MHV-JHM neutralization in murine milk. Offspring of transgenic and control mice were challenged with a lethal dose of MHV-JHM. Litters suckling nontransgenic dams succumbed to fatal encephalitis, whereas litters suckling transgenic dams were fully protected against challenge, irrespective of whether they were transgenic. This demonstrates that a single neutralizing antibody expressed in the milk of transgenic mice is sufficient to completely protect suckling offspring against MHV-JHM-induced encephalitis.
Animals↗
Multiple copies of beta-lactoglobulin promoter do not function as LCR.
Increasing the number of transcription factor binding sites within a construct can enhance expression. In an attempt to create a synthetic locus control region for mammary expression, we have generated beta-lactoglobulin-reporter constructs with multiple copies of the cluster of transcription sites normally located within the proximal promoter. These constructs were functionally tested by stable transfection of mammary epithelial cells in vitro and in transgenic mice in vivo. Rather than enhancing expression, multimerisation of the promoter region acted neither in vivo nor in vitro to enhance expression. Indeed, its presence reduced expression. This failure to enhance expression was reflected in the inability of this region to form a DNaseI hypersensitive site autonomously in mammary chromatin in vivo. It is implicit from our study that not all combinations of transcription factor binding sites will enhance transcription.
Animals↗
Matrix attachment region regulates basal beta-lactoglobulin transgene expression.
Nuclear matrix attachment regions (MAR) have been implicated in the regulation of gene expression. We have identified a region within the proximal 3'-flanking sequences of the ovine beta-lactoglobulin (betalg) gene that interacts with the nuclear matrix in vitro. No equivalent region was detected in the 5' flanking region. We have investigated the role of this element in regulating betalg expression in vitro and in vivo. Removal of the MAR did not affect the frequency of betalg transgene expression at the mRNA level, but betalg transgenes that lacked the MAR were expressed at a lower level than wild-type betalg transgenes. In neither in-vitro HC11 transfection experiments nor transgenic mice was hormonal induction of betalg expression significantly affected by MAR removal. Nuclear run-on analysis demonstrated that the impaired basal expression of betalg transgene loci lacking the MAR was due to a reduced transcription rate. Thus, the single MAR enhances the basal transcriptional potential of the betalg gene.
Animals↗
Nucleosome organisation of the beta-lactoglobulin gene. Transcription complex formation.
In most mammals, the major whey protein beta-lactoglobulin (beta lg) represents a marker for tissue-specific, temporally regulated gene expression in the mammary gland. Prolactin, the major lactogenic stimulus which activates beta lg expression acts through a cytoplasmic signalling cascade ending in the activation of the transcription factor STAT5. Although much is known about the complexities of this signalling cascade, little is known about how this transcription factor functions within the context of chromatin. Using DNaseI as a probe of chromatin structure we have identified temporally regulated elements within the ovine beta lg gene domain. The appearance of these hypersensitive sites accompanies changes in expression state of the beta lg gene. Changes in DNaseI hypersensitivity at the proximal promoter region, while reflecting STAT5 activation, is not dependent upon STAT5 interaction at this site. We have mapped the nucleosome positions over the entire beta lg gene, both in vitro using the monomer extension assay and in vivo using cuprous phenanthroline to probe for nucleosome-linker positions. The specific positioning pattern detected, which reflects strong sequence-directed positioning over the proximal promoter, complement the STAT5 consensus sites within this region. The comparison of both the functional and chromatin data enables a model for beta lg gene transcription to be developed.
Animals↗
Insulin-like growth factor binding protein-5 (IGFBP-5) potentially regulates programmed cell death and plasminogen activation in the mammary gland.
This study aims to investigate the mechanism by which prolactin and GH interact to maintain mammary epithelial cell function in the rat. IGF-I is an important survival factor for the mammary gland and we have demonstrated that the effects of GH and prolactin involve IGF-I. GH acts by increasing IGF-I whilst prolactin acts by inhibiting the expression of IGFBP-5 from the mammary epithelium. During mammary involution, when serum prolactin levels decline, IGFBP-5 expression is dramatically upregulated and it binds with high affinity to IGF-I preventing IGF-I interaction with the IGF-receptor and thus leading to epithelial cell apoptosis. We have identified a specific interaction of IGFBP-5 with alpha s2-casein. This milk protein has also been shown to bind plasminogen and its activator tissue-type plasminogen activator (tPA) leading to enhanced conversion of plasminogen to plasmin. Plasmin is an important initiator of re-modelling of the extracellular matrix during mammary involution. A potential interaction between the cell death and extracellular matrix remodelling is evident from the observation that IGFBP-5 binds to plasminogen activator inhibitor-I (PAI-1). We thus hypothesized that IGFBP-5 could activate cell death by sequestration of IGF-I and activate plasminogen cleavage by sequestering PAI-1. In support of this hypothesis we have shown that both prolactin and GH inhibit tPA activity and plasminogen activation in the involuting mammary gland. Our results suggest that GH and prolactin inhibit cell death and ECM remodelling via the IGF-axis and also indicate a novel role for the milk protein alpha s2-casein in this process. We have now established lines of transgenic mice expressing IGFBP-5 on the beta-lactoglobulin promoter to explore its function in greater detail.
Animals↗
Chromatin structures of goat and sheep beta-lactoglobulin gene differ.
Different levels of the major milk protein beta-lactoglobulin are found in evolutionarily related ruminant species: with sheep milk containing as much as three times the concentration in goat milk. In an attempt to understand why these differences exist, we have characterised, using DNaseI as a probe of structure, the chromatin surrounding the goat beta-lactoglobulin promoter and compared it to that of the sheep homologue. The goat gene displays a mammary-specific chromatin pattern, which is reformed on expressing goat beta-lactoglobulin transgenes. This implies that this chromatin structure is sequence dependent and suggests that it plays a role in regulating beta-lactoglobulin gene expression. This pattern differs from that seen on the ovine beta-lactoglobulin gene in lactating sheep mammary chromatin. Thus, even between highly related species, the transcriptional mechanisms regulating activity of a gene can differ.
Animals↗
Temporal profiles of appearance of DNase I hypersensitive sites associated with the ovine beta-lactoglobulin gene differ in sheep and transgenic mice.
The ovine milk protein beta-lactoglobulin is expressed in a distinct temporal pattern during lactogenesis. This expression pattern is reflected in the temporal profile of appearance of DNase I hypersensitive sites (HS) associated with the beta-lactoglobulin gene in the mammary gland. Specifically, HSIV and HSV are present prior to the first major increase in expression, which occurs at mid-pregnancy, while HSI displays the converse profile, being detected after mid-pregnancy and during lactation. The extent of DNase I digestion at HSIII, encompassing the promoter region, reflects the level of beta-lactoglobulin expression. In transgenic mouse mammary chromatin, beta-lactoglobulin transgenes display the same set of DNase I hypersensitive sites as in sheep mammary chromatin. The temporal profile, however, differs from that seen in sheep: notably, HSIV and HSV are detected during lactation. The fact that beta-lactoglobulin transgenes lacking HSIV and HSV are expressed but display a reduced transcription rate per integrated copy is compatible with a functional role for these regions. This suggests that HSIV and HSV may increase the likelihood of high-level transgene expression.
Animals↗
Rescue of an MMTV transgene by co-integration reveals novel locus control properties of the ovine beta-lactoglobulin gene that confer locus commitment to heterogeneous tissues.
In an attempt to enhance the frequency and level of expression of a poor-performing MMTV-driven transgene, we co-integrated this construct with the ovine beta-lactoglobulin (BLG) gene in transgenic mice. Seven lines of transgenic mice possessing co-integrated BLG and MMTV-RZ5 transgenes were compared with 12 lines of mice that possessed only the MMTV-RZ5 construct. Co-integration enhanced the frequency of expression in the mammary gland from two out of 12 lines for the MMTV-RZ5 transgene alone, to five out of seven when co-integrated with BLG. Surprisingly, co-integration also resulted in co-expression of the two transgenes in the salivary gland, lung and spleen in addition to the mammary gland. Furthermore, both transgenes were expressed in virgin animals, and throughout pregnancy and lactation, suggesting that the developmental regulation of the locus follows that of the MMTV-promoter. These findings represent a novel locus control property of the ovine BLG gene that confers commitment of the locus to the mammary gland, but also to a range of heterogeneous tissues possibly defined by the second promoter at the locus.
Animals↗
Transgenic mice secreting coronavirus neutralizing antibodies into the milk.
Ten lines of transgenic mice secreting transmissible gastroenteritis coronavirus (TGEV) neutralizing recombinant monoclonal antibodies (rMAbs) into the milk were generated. The rMAb light- and heavy-chain genes were assembled by fusing the genes encoding the variable modules of the murine MAb 6A.C3, which binds an interspecies conserved coronavirus epitope essential for virus infectivity, and a constant module from a porcine myeloma with the immunoglobulin A (IgA) isotype. The chimeric antibody led to dimer formation in the presence of J chain. The neutralization specific activity of the recombinant antibody produced in transiently or stably transformed cells was 50-fold higher than that of a monomeric rMAb with the IgG1 isotype and an identical binding site. This rMAb had titers of up to 10(4) by radioimmunoassay (RIA) and neutralized virus infectivity up to 10(4)-fold. Of 23 transgenic mice, 17 integrated both light and heavy chains, and at least 10 of them transmitted both genes to the progeny, leading to 100% of animals secreting functional TGEV neutralizing antibody during lactation. Selected mice produced milk with TGEV-specific antibody titers higher than 10(6) as determined by RIA, neutralized virus infectivity by 10(6)-fold, and produced up to 6 mg of antibody per ml. Antibody expression levels were transgene copy number independent and integration site dependent. Comicroinjection of the genomic beta-lactoglobulin gene with rMAb light- and heavy-chain genes led to the generation of transgenic mice carrying the three transgenes. The highest antibody titers were produced by transgenic mice that had integrated the antibody and beta-lactoglobulin genes, although the number of transgenic animals generated does not allow a definitive conclusion on the enhancing effect of beta-lactoglobulin cointegration. This approach may lead to the generation of transgenic animals providing lactogenic immunity to their progeny against enteric pathogens.
Amino Acid Sequence↗
Expression of c-myc is down-regulated as mouse mammary epithelial cells become confluent.
We have investigated the expression profile of c-myc in the mammary gland. During pregnancy when the gland is actively growing c-myc mRNA was present, while in the differentiated lactating gland no c-myc mRNA was detected. This correspondence between the differentiation state and c-myc mRNA levels in the mouse mammary gland in vivo was paralleled by HC11 mouse mammary epithelial cells in vitro. Firstly, the endogenous c-myc gene was suppressed in confluent compared to growing HC11 cells. In addition, treating the cells with lactogenic hormones did not induce c-myc expression. Secondly, a stably transfected c-myc-CAT reporter construct was similarly down-regulated. Furthermore, using this transfection model, we demonstrate that the mechanism(s) involved in regulating c-myc expression must act through the P1 and P2 core promoter and exon 1. Finally, we demonstrate that suppression of c-myc expression occurs when HC11 cells growth-arrest as they become confluent.
Animals↗
Intronic sequences modulate the sensitivity of beta-lactoglobulin transgenes to position effects.
We have analysed the expression of beta-lactoglobulin (BLG) gene constructs with combinations of introns deleted to further define the role of intronic regions in directing position-independent mammary expression of BLG transgenes. Intron removal had no obvious effect on hormonal induction of BLG expression in vitro but dramatically reduced expression in vivo, in that removal of intron pairs always resulted in a proportion of the transgenic lines generated failing to express the transgene in the mammary gland. Position-dependent expression was seen for all intron-deleted transgenes regardless of which introns were removed and the ability of the intron-deleted transgenes to be expressed bore no relationship to transgene copy number. Thus, intron removal per se increases the sensitivity of BLG transgenes to position effects.
Animals↗
Truncated beta-lactoglobulin transgenes are expressed in the kidney.
The major milk whey protein of ruminants is beta-lactoglobulin. Ovine beta-lactoglobulin-encoding gene expression is restricted to the sheep mammary gland. This report describes the expression profile of a truncated beta-lactoglobulin transgene which, although not expressed in the mammary gland, is expressed in the kidney in the majority of lines generated. The high frequency of ectopic kidney expression may relate to the ability of the larger beta-lactoglobulin transgenes to be expressed in a position-independent manner in the mammary gland of transgenic mice.
Animals↗
Comparable processing of beta-lactoglobulin pre-mRNA in cell culture and transgenic mouse models.
Eukaryotic pre-mRNAs undergo a variety of post-transcriptional modifications, including the removal of intronic sequences by splicing, leading to creation of a functional mRNA. We have compared the processing of transcripts generated from ovine beta-lactoglobulin gene constructs in stably transfected cells and in transgenic mice. In both the in vitro and in vivo model systems the removal of the middle two introns resulted in the inefficient splicing of the downstream, terminal intron. This intron-containing transcript was detected in the cytoplasmic RNA fraction. Thus, the initial in vitro analysis in cell lines of minigene constructs destined for expression in transgenic animals may provide a rapid and reliable indicator of the processing efficiency of the pre-mRNA produced by the construct in vivo. This is in contrast to the apparent limitations of in vitro systems in the analysis of transcription regulatory elements required for transgene expression.
Animals↗
Hormonal influences on beta-lactoglobulin transgene expression inferred from chromatin structure.
The major milk whey protein of ruminants is beta-lactoglobulin. Transgenic mice which carry genomic fragments of ovine beta-lactoglobulin express the transgene at high levels in the mammary gland. Using DNaseI as a probe for transcription complex formation in chromatin, the temporal induction pattern of beta-lactoglobulin in transgenic mice has been addressed and compared to the known hormonal profiles during pregnancy. Prior to the 9th day of pregnancy no obvious hypersensitivity to DNaseI digestion at the beta-lactoglobulin promoter was evident. From the 9th day of pregnancy through to lactation, the beta-lactoglobulin promoter displays DNaseI hypersensitivity. These results support the hypothesis that placental lactogens are the major lactogenic influence from mid-pregnancy to parturition.
Animals↗
Stat5 as a target for regulation by extracellular matrix.
Transcription of tissue-specific genes in mammary gland requires signals from both prolactin and basement membrane. Here we address the mechanism by which this specialized extracellular matrix regulates transcription. Using mammary cell cultures derived from transgenic mice harboring the ovine beta-lactoglobulin gene, we show that either a basement membrane extract, or purified laminin-1, induced high levels of beta-lactoglobulin synthesis. It is known that prolactin signals through Stat5 (signal transducer and activator of transcription). This transcription factor interacts with gamma-interferon activation site-related motifs within the beta-lactoglobulin promoter, which we show are required for matrix dependence of beta-lactoglobulin expression. The DNA binding activity of Stat5 was present only in extracts of mammary cells cultured on basement membrane, indicating that the activation state of Stat5 is regulated by the type of substratum the cell encounters. Thus, basement membrane controls transcription of milk protein genes through the Stat5-mediated prolactin signaling pathway, providing a molecular explanation for previous studies implicating extracellular matrix in the control of mammary differentiation.
Animals↗
Regulation of ovine beta-lactoglobulin gene expression during the first stage of lactogenesis.
In the lactating mammary gland, expression of the ovine beta-lactoglobulin gene correlates with the presence of a strong DNaseI hypersensitive site encompassing the promoter region. At this stage of lactogenesis, prolactin is required for optimal expression. Using DNaseI as a probe for formation of a transcription complex on the beta-lactoglobulin promoter, the temporal pattern of ovine beta-lactoglobulin expression during pregnancy has been addressed. The appearance of nuclease hypersensitivity during pregnancy correlates with rising levels of placental lactogen which suggests that this hormone may be the stimulus for expression during the first stage of lactogenesis. This raises the possibility that a different signalling pathway to that present in the lactation functions during pregnancy.
Animals↗
Ectopic expression of beta-lactoglobulin transgenes.
Genomic constructs comprising the ovine beta-lactoglobulin gene are expressed in a position-independent manner in the mammary gland of transgenic mice. In some lines however, constitutive low-level transgene expression was detected in all other tissues. This ectopic expression presumably represents a position-dependent phenomenon since it was observed in only a proportion (40%) of the lines generated. Different lines of BLG transgenic mice displayed similar temporal patterns of ectopic expression. This pattern differed from that of BLG in the mammary gland. These data imply that the DNA elements that direct position-independent expression of beta-lactoglobulin transgenes in the mammary gland do not have the ability to insulate them from position effects in other tissues. Furthermore, the relatively high frequency and constitutive nature of ectopic expression suggests that transgene integration may not be totally random.
Animals↗