Expression of the human granulocyte–macrophage colony stimulating factor (hGM-CSF) gene under control of the 5′-regulatory sequence of the goat alpha-S1-casein gene with and without a MAR element in transgenic mice

Expression of the human granulocyte–macrophage colony-stimulating factor (hGM-CSF) gene under the control of the 5′-regulatory sequence of the goat alpha-S1-casein gene with and without a matrix attachment region (MAR) element from the Drosophila histone 1 gene was studied in four and eight transgenic mouse lines, respectively. Of the four transgenic lines carrying the transgene without MAR, three had correct tissues-specific expression of the hGM-CSF gene in the mammary gland only and no signs of cell mosaicism. The concentration of hGM-CSF in the milk of transgenic females varied from 1.9 to 14 μg/ml. One line presented hGM-CSF in the blood serum, indicating ectopic expression. The values of secretion of hGM-CSF in milk of 6 transgenic lines carrying the transgene with MAR varied from 0.05 to 0.7 μg/ml, and two of these did not express hGM-CSF. Three of the four examined animals from lines of this group showed ectopic expression of the hGM-CSF gene, as determined by RT-PCR and immunofluorescence analyses, as well as the presence of hGM-CSF in the blood serum. Mosaic expression of the hGM-CSF gene in mammary epithelial cells was specific to all examined transgenic mice carrying the transgene with MAR but was never observed in the transgenic mice without MAR. The mosaic expression was not dependent on transgene copy number. Thus, the expected “protective or enhancer effect” from the MAR element on the hGM-CSF gene expression was not observed.     

Bovine αs1-casein gene sequences direct high level expression of human granulocyte-macrophage colony-stimulating factor in the milk of transgenic mice

The generation is reported of transgenic mice expressing human granulocyte-macrophage colony-stimulating factor (GM-CSF) or human erythropoietin (EPO) under the control of bovine s1- casein regulatory sequences. GM-CSF expression was specific to the mammary gland, and levels of human GM-CSF in transgenic mouse milk were in the range of mg ml–1. The specific activity of the milk GM-CSF was similar to that of the recombinant protein produced in Escherichia coli, and the glycosylation-derived size heterogeneity corresponded to that of the native human protein. In spite of the identical bovine regulatory sequences of the fusion genes, the levels of human EPO in transgenic mouse milk were 103--106 times lower than those of GM-CSF, ranging from 0.003 to 3 g ml–1. There appeared to be a positive correlation between the amount of EPO in the milk of lactating females and blood haematocrit values. In view of this, other type of constructs should be used to achieve more efficient EPO expression and to circumvent concomitantly-occurring adverse effects. In contrast, the high-level production of recombinant GM-CSF, its resemblance to the native mammalian protein, and mild adverse consequences of transgene expression imply that the current construct could be used for generation of larger GM-CSF transgenic anim als to produce this protein in quantities sufficient for therapeutic purposes     

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

Transgenic Research -     

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 ratelimiting for the transport of dIgA across the epithelial cells into the secretions. We address this key issue by generating transgenic mice overexpressing 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 _s1casein 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 2.7mg/ml during midlactation. These levels are 10–270 times higher than wildtype SC levels (0.01mg/ml).     

A hybrid bovine ß-casein/bGH gene directs transgene expression to the lung and mammary gland of transgenic mice

We investigated spatial and temporal expression of bGH controlled by two different sizes (1.8kb and 15kb) of 5-flanking sequences of the bovine ß-casein in transgenic mice. In the 1.8-kb promoter-containing mice, bGH expression was specifically confined to lung and mammary gland at lactation. While mammary gland expression was highly variable depending on the lines, lung expression was relatively constant with a high level in most lines. Moreover, this dual-tissue specificity of bGH expression was consistently retained in all of the 15kb-promoter-containing mice, although a low ectopic expression was sometimes detected in salivary gland or brain. During mammary gland development in the 1.8-kb promoter-containing mice was mammary gland expression first detected at lactation, following the bovine rather than murine pattern of ß-casein expression. In contrast, lung expression was almost constant regardless of mammary gland developmental state or sex. Therefore, it can be concluded that a combination of the bovine ß-casein promoter and bGH gene directs a distinct dual-tissue specific bGH expression with different regulatory mechanisms between mammary gland and lung and as little as 1.8-kb promoter is sufficient for the proper regulation of the bovine ß-casein gene in mammary gland.     

A new β-lactoglobulin-based vector targets luciferase cDNA expression to the mammary gland of transgenic mice

A -lactoglobulin (BLG)/luciferase gene vector (p907), composed of a luciferase intronless gene inserted between the second and sixth BLG exons was constructed. Stable transfections of CID-9 cells with this vector, as well as with a series of additional vectors, were performed to define regulatory regions within the BLG sequence, and the contribution of the SV40 polyadenylation (PA) site to luciferase expression. A relatively low level of luciferase activity was supported by vector p907. It was partially rescued by vector p906, in which the BLG 3 region, downstream of the luciferase cDNA, was replaced with the SV40 PA site. Flanking the SV40 region of vector p906, at its 3 end, with BLG sequences of exon 6/intron 6/exon 7 and the 3 region of the gene resulted in vector p904. This vector supported the highest luciferase activity, 10 times or 2.5 times higher than that measured in cells transfected with vectors p907 and p906, respectively. The induced activity supported by vector p904 is attributed to interaction between the SV40 PA site and elements of the distal part of the BLG 3 flanking sequences. The BLG 5 regulatory region of vector p904 encompasses a 3-kb promoter sequences. Deletion of 935 bp of its proximal end resulted in a 60% decrease in luciferase activity. Reduced activity was also seen with vector p915 lacking sequences of exon 1/intron 1/exon 2. This decrease could not be rescued with heterologous sequences of insulin intron 1, inserted upstream of the luciferase cDNA. Two sets of transgenic mice carrying vectors p907 and p904 were generated. Vector p907 supported only marginal luciferase activity in the mammary gland of all transgenic mice tested and luciferase RNA could not be detected by northern analysis. In contrast, 50% of the transgenic mice carrying vector p904 expressed luciferase RNA in the mammary gland and tissue-specific, hormonal-dependent activity was determined. However, the new p904 vector was not able to insulate the transgene from surrounding host DNA sequences, as reflected by its copy number-independent manner of expression. Nevertheless, vector p904 may represent a valuable tool for the expression of cDNAs in the mammary gland of transgenic animals.     

β-Glucanase specific expression in the parotid gland of transgenic mice

The feasibility of using the pig parotid secretory protein promoter to drive the β-glucanase transgene expression in mouse parotid glands was examined in this study. The parotid gland-specific vector expressing β-glucanase gene (GLU, from Paenibacillus polymyxa CP7) was constructed. Transgenic mice were produced by the pronuclear microinjection. Both PCR and Southern blot analysis showed that the mice carried the β-glucanase gene and the β-glucanase gene could be stably inherited. Furthermore, RT-PCR and northern blot analysis indicated that it was specifically expressed in the parotid. The β-glucanase activity in the saliva was found to be 0.18 U/mL. After feeding a diet containing 2 % β-glucan, the average daily gain of transgenic was significantly higher than non-transgenic mice. The crude protein and crude fat concentration in faeces of transgenic mice were significantly reduced compared with that of the non-transgenic mice. These results suggest that the successful expression of foreign β-glucanase in the animal parotid would offer a promising biological approach to reduce the anti-nutritional effect of β-glucans in feed.     

Analysis of developmental switching in transgenic mice with 5′ and 3′ deletions in the human β globin gene

Our interest in thecis-acting elements that promote the up-regulation of the globin gene has led to a systematic deletion analysis of portions of the globin gene in the context of the HS2 and globin gene using transgenic mice. In constructs that delete the 5 region to only 265 bp, high-level erythroid-specific expression was observed. Further deletion to 122 bp, however, results in significantly reduced expression levels A substitution of a minilocus control region for the single HS2 site was also produced, resulting in increased globin expression over that seen with the HS2 alone. These results are consistent with the presence of an enhancer-like element between –122 and –265. In addition, a construct in which the entire globin gene promoter was replaced by a thymidine kinase promoter was tested. Interestingly, no expression was detected in these transgenic mice. This may indicate the requirement for an erythroid-specific promoter to drive this gene. Finally, the 3 region of the globin gene was deleted in order to examine the effect of a previously defined 3 enhancer region. With deletion of this region, the expression of the human globin gene in transgenic mice is unchanged relative to the parental constructs.     

Restricted tissue-specific but correct developmental expression mediated by a short human α1AT promoter fragment in transgenic mice

The tissue-specific and developmental pattern of expression controlled by the proximal promoter (position –348 to+15) derived from the human -1-antitrypsin (h1AT) gene was studied in transgenic mice. The short promoter segment was linked to the chloramphenicol acetyltransferase (CAT) reporter gene. The transgene showed highly specific expression in the liver and the correct developmental pattern of regulation. Interestingly, this short promoter targets expression to the liver with a greater specificity than that reported for larger 1AT promoter fragments.     

DNA polymorphisms in the 5′-flanking region of the HLA-DQA1 gene

The HLA-DQA1 gene exhibits haplotype-specific restriction fragment polymorphisms due to DNA rearrangements. We found that some of these polymorphisms extend into the 5 flanking region of the gene and are distinct from other HLA-DQA1 related DNA polymorphisms so far reported. Sequencing of genomic DNA subclones derived from the 5 flanking region of HLA-DQA1 showed the presence, in a DR4 haplotype, of two repetitive elements of the Alu family, oriented in opposite directions and bracketing an approximately 3 kilobase region immediately adjacent to the promoter of the gene. When DNAs extracted from several cell lines were analyzed by genomic hybridization using single-copy probes relative to these intervening sequences, polymorphisms were observed. No structural alterations of the gene immediately outside the DNA portion delimited by the two Alu elements were observed, thus suggesting that polymorphisms of the 5 end of HLA-DQA1 may be limited to the intervening region between the two Alu repeats. The latter includes upstream regulatory elements controlling the expression of the genes. The possibility that the structure of the DNA in this region may influence the regulation of HLA-DQA1 gene expression in different haplotypes is discussed.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M72411. Address correspondence and offprint requests to: J. Guardiola.     

A mWAP–hLF hybrid gene locus gave extremely high level expression of human lactoferrin in the milk of transgenic mice

The production of pharmaceuticals by current mammary gland bioreactor techniques is limited by the low expression level of foreign proteins. We describe here a novel method that solves this problem. A successive three-step gap-repair strategy was developed to replace the genomic coding sequence in mouse whey acidic protein (mWAP) gene locus with that of human lactoferrin (hLF) precisely from the start code to the end code. A 50-kb mWAP–hLF hybrid gene locus was constructed, and corresponding transgenic mice were generated. An extremely high-level expression of rhLF in the milk was demonstrated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot. The expression level ranged from 16.7 to 29.8 g/l among five transgenic lines, as indicated by the ELISA assay. Importantly, the expressed rhLF maintained the same antibacterial activity as the native hLF. Our strategy can very likely also be used for the efficient expression of other valuable pharmaceutical proteins. G. Shi and H. Chen contributed equally to this work.     

Expression of a bovine κ-CN cDNA in the mammary gland of transgenic mice utilizing a genomic milk protein gene as an expression cassette

Transgenic mice were produced by microinjection of a DNA construct composed of the bovine -casein (-CN) cDNA under the control of the goat -CN 5 promoter elements and 3 flanking regions into pronuclear-stage embryos. The gene construct targeted the expression of bovine -CN RNA to the mammary gland and secretion of bovine -CN in the milk. In the three lines studied (BC-7, BC-31 and BC-67) the transgene was stably integrated and propagated as a Mendelian locus. Expression of the bovine protein in lactating mice from the three transgenic lines was demonstrated by northern and western blots. In ten different tissues analysed by northern blotting, expression was confined to the mammary gland of lactating transgenic mice from line BC-7, with low-level expression also observed in the salivary gland of lines BC-31 and BC-67. Transgene expression in the mammary gland paralleled normal casein gene expression during lactation and was not observed in virgin females. The level of bovine -CN mRNA expression on day 10 of lactation in hemizygous transgenic females in relation to endogenous mRNA of whey acid protein (WAP) gene expression was 14%, 69% and 127% in lines BC-7, BC-31 and BC-67, respectively. No association between transgene copy number and expression was observed. The bovine -CN concentration in milk on day 10 of lactation ranged from 0.94 to 3.85 mg of protein per ml of milk. The bovine -CN expressed in mouse milk had the same molecular mass and immunoactivity with polyclonal antibodies as did -CN from bovine milk. A high degree of variation in the production of bovine -CN within each of the transgenic lines was observed.     

Multiple roles of the coding sequence 5′ end in gene expression regulation

The codon composition of the coding sequence''s (ORF) 5′ end first few dozen codons is known to be distinct to that of the rest of the ORF. Various explanations for the unusual codon distribution in this region have been proposed in recent years, and include, among others, novel regulatory mechanisms of translation initiation and elongation. However, due to the fact that many overlapping regulatory signals are suggested to be associated with this relatively short region, its research is challenging. Here, we review the currently known signals that appear in this region, the theories related to the way they regulate translation and affect the organismal fitness, and the debates they provoke.     

Evaluation of seed storage-protein gene 5′ untranslated regions in enhancing gene expression in transgenic rice seed

5′ untranslated regions (UTRs) are important sequence elements that modulate the expression of genes. Using the β-glucuronidase (GUS) reporter gene driven by the GluC promoter for the rice-seed storage-protein glutelin, we evaluated the potential of the 5′-UTRs of six seed storage-protein genes in enhancing the expression levels of the foreign gene in stable transgenic rice lines. All of the 5′-UTRs significantly enhanced the expression level of the GluC promoter without altering its expression pattern. The 5′-UTRs of Glb-1 and GluA-1 increased the expression of GUS by about 3.36- and 3.11-fold, respectively. The two 5′-UTRs downstream of the Glb-1, OsAct2 and CMV35S promoters also increased GUS expression level in stable transgenic rice lines or in transient expression protoplasts. Therefore, the enhancements were independent of the promoter sequence. Real-time quantitative RT-PCR analysis showed that the increase in protein production was not accompanied by alteration in mRNA levels, which suggests that the enhancements were due to increasing the translational efficiencies of the mRNA. The 5′-UTRs of Glb-1 and GluA-1, when combined with strong promoters, might be ideal candidates for high production of recombinant proteins in rice seeds.     

A 470 bp WAP-promoter fragment confers lactation independent,progesterone regulated mammary-specific gene expression in transgenic mice

The ability of a 470 bp sub-fragment of the murine whey acidic protein (WAP) promoter in the context of a retroviral expression plasmid to direct gene expression to mammary epithelial cells was analysed in a number of independent transgenic mouse lines. In contrast to previous findings with the genuine 2.5 kb promoter fragment, our studies revealed a highly mammary gland-specific expression detectable only in non-lactating animals. This suggested a mainly progesterone-regulated activity of the short fragment. Therefore, transgene expression was examined in the progesterone-determined estrous cycle and during pregnancy. In accordance with in vitrodata from stably transfected cell lines, in both situations expression was upregulated at stages associated with high progesterone levels. Taken together these data provide deeper insight into WAP-promoter regulation and stress the usefulness of the shortened fragment for a lactation independent mammary-targeted expression.     

The 5′-UTR intron of the midgut-specific BmAPN4 gene affects the level and location of expression in transgenic silkworms

Introns are important for regulating gene expression. BmAPN4, which has a 5′-UTR upstream intron (5UI), is specifically expressed in the entire silkworm midgut. In our previous study, the promoter region upstream of the 5UI of BmAPN4 was cloned and identified as the P3 promoter (P3P) with activity only in the anterior midgut. In this study, the sequence consisting of the P3P and the 5UI was cloned and named as P3P+5UI. A transgenic vector was constructed in which EGFP was controlled by P3P+5UI. Transgenic P3+5UI silkworms were generated by embryo microinjection. RT-PCR showed P3P+5UI activity throughout the larval stage. Intense green fluorescence was seen only in the entire midgut of P3+5UI silkworms and expression was confirmed by RT-PCR. qPCR revealed that expression of EGFP in the anterior midgut of P3+5UI silkworms was 64% higher than in P3 silkworms, indicating the 5UI sustained intron-mediated enhancement of gene expression. These results suggested that the BmAPN4 5UI affected the level and site of expression. The 5UI was cloned and added behind P2P, another specific promoter with activity only in the anterior midgut of silkworm, to construct the P2P+5UI and transgenic P2+5UI silkworms. Expression patterns were the same for P2P+5UI and P2P, suggesting that the 5UI of BmAPN4 did not affect P2P. This study found that the BmAPN4 5UI affected the amount and location of gene expression. Its influence appeared to be dependent on a specific promoter.