Tissue-specific expression in the salivary glands of transgenic mice.

Using a DNA construct, named Lama, derived from the murine parotid secretory protein (PSP) gene, we have obtained salivary gland specific gene expression in transgenic mice. Lama is a PSP minigene and allows analysis of the PSP gene 5' regulatory region by transgenesis. We show here that the regulatory region included in Lama with 4.6 kb of 5' flanking sequence is sufficient to direct expression specifically to the salivary glands. The expression level in the parotid gland is only about one percent of the PSP mRNA level, while that of the sublingual gland is near the PSP mRNA level. This suggests significant differences in the PSP gene regulation in the two glands. In addition, Lama is a secretory expression vector in which cDNAs or genomic fragments can be inserted. We demonstrate that the Lama construct can direct the expression of a heterologous cDNA encoding the C-terminal peptide of human factor VIII to salivary glands and that the corresponding peptide is secreted into saliva.     

High-level salivary gland expression in transgenic mice

A 7.1 kb mini-gene construct containing cloned DNA from the murine parotid secretory protein (PSP) gene with 6.2 kb of the promoter, has previously been shown to direct specific mRNA expression to the salivary glands in transgenic mice. However, the level of transgene expression in the parotid gland was only a few percent of the endogenous level. This indicated that elements necessary for high-level expression are still to be found. In this study, we have searched for such regulatory elements in additional flanking regions by using a 25 kb clonedPsp ^b fragment containing the complete structural gene, 11.4 kb of 5-flanking sequence, and 2.5 kb 3-flanking sequence as a transgene. To distinguish the expression of the transgene from that of the endogenous gene, we took advantage of an allelic difference, using an oligonucleotide that recognized the mRNA fromPsp ^b and the transgene but not that from the other allele,Psp ^a . The expression of the transgene was examined in animals homozygous forPsp ^a . Three independent integrations all exhibited a level of parotid-gland-specific expression that corresponded to that of the endogenous gene. Thus, sequences responsible for this high-level PSP mRNA expression are situated within the genomic DNA of the transgene.     

Synthesis and secretion of the mouse whey acidic protein in transgenic sheep

The synthesis of foreign proteins can be targeted to the mammary gland of transgenic animals, thus permitting commercial purification of otherwise unavailable proteins from milk. Genetic regulatory elements from the mouse whey acidic protein (WAP) gene have been used successfully to direct expression of transgenes to the mammary gland of mice, goats and pigs. To extend the practical usefulness of WAP promoter-driven fusion genes and further characterize WAP expression in heterologous species, we introduced a 6.8 kb DNA fragment containing the genomic form of the mouse WAP gene into sheep zygotes. Two lines of transgenic sheep were produced. The transgene was expressed in mammary tissue of both lines and intact WAP was secreted into milk at concentrations estimated to range from 100 to 500 mg/litre. Ectopic WAP gene expression was found in salivary gland, spleen, liver, lung, heart muscle, kidney and bone marrow of one founder ewe. WAP RNA was not detected in skeletal muscle and intestine. These data suggest that unlike pigs, sheep may possess nuclear factors in a variety of tissues that interact with WAP regulatory sequences. Though the data presented are based on only two lines, these findings suggest WAP regulatory sequences may not be suitable as control elements for transgenes in sheep bioreactors.     

Expression of hGM-CSF in silk glands of transgenic silkworms using gene targeting vector

The silk gland of the silkworm is a highly specialized organ that has the wonderful ability to synthesize and secrete silk protein. To express human granucyto-macrophage colony-stimulating factor (hGM-CSF) in the posterior silk glands of gene-targeted silkworms, a targeting vector pSK-FibL-L-A3GFP-PH-GMCSF-LPA-FibL-R was constructed, harboring a 1.2 kb portion of the left homogenous arm (FibL-L), a 0.5 kb portion of the right homogenous arm (FibL-R), fibroin H-chain-promoter-driven hGM-CSF and silkworm actin 3-promoter-driven gfp. The targeting vector was then introduced into the eggs of silkworm, and the transgenic silkworms were verified by PCR and DNA hybridization after being screened for the gfp gene. Western blotting analysis using an antibody against hGM-CSF demonstrated a specific band with a molecular weight of 22 kD in the silk glands of the G3 generation transgenic silkworms. The level of expression of hGM-CSF in the posterior silk glands of the G3 generation transgenic silkworms was approximately 2.70 ng/g of freeze-dried powdered posterior silk gland. These results showed that the heterologous gene could be introduced into the silkworm genome and expressed successfully. Further more, the exogenous genes existing in the G5 transgenic silkworm identified by PCR confirmed its integration stability. In addition, the silk glands containing expressed hGM-CSF performed the function of significantly increasing leukocyte count of CY-treated mice in a time-and-dose-dependent manner.     

Establishment of La-tPA/G-CSF dual transgenic mice and expression in their mammary gland

Expression vectors of human granulocyte colony stimulating factor (G-CSG) and long acting tissue plasminogen activator (La-tPA) in mammary gland were constructed using promoters of mouse whey acid protein gene (WAP) and sheep β-lactoglobulin gene (BLG) with sizes of 2.6 and 5 kb respectively. Two kinds of transgenic mice of G-CSF and La-tPA were produced with microinjection. The expression of G-CSF and La-tPA was achieved in mammary glands of transgenic mice, respectively. In order to establish dual transgenic mice of La-tPA /G-CSF, transgenic mice carrying G-CSF and La-tPA gene characterized with specific expression in mammary gland were mated. La-tPA/G-CSF dual transgenic mice were screened out from the hybrid offspring by Once-PCR. The co-expression of La-tPA and G-CSF in mammary gland of the dual transgenic mice was confirmed by the milk assayed and Northern blot analysis. Some parameters about the dual transgenic mice indicated that there were fewer litters than that of normal mice. The ratio of dual transgenes was 46.1% in F1 generation, and offspring’s sex ratio was normal. Hence a dual transgenic mouse model was established for the study of co-expression foreign proteins in mammary gland.     

Establishment of La-tPA/G-CSF dual transgenic mice and expression in their mammary gland

Expression vectors of human granulocyte colony stimulating factor (G-CSG) and long acting tissue plasminogen activator (La-tPA) in mammary gland were constructed using promoters of mouse whey acid protein gene (WAP) and sheep β-lactoglobulin gene (BLG) with sizes of 2.6 and 5 kb respectively. Two kinds of transgenic mice of G-CSF and La-tPA were produced with microinjection. The expression of G-CSF and La-tPA was achieved in mammary glands of transgenic mice, respectively. In order to establish dual transgenic mice of La-tPA/G-CSF, transgenic mice carrying G-CSF and La-tPA gene characterized with specific expression in mammary gland were mated. La-tPA/G-CSF dual transgenic mice were screened out from the hybrid offspring by Once-PCR. The co-expression of La-tPA and G-CSF in mammary gland of the dual transgenic mice was confirmed by the milk assayed and Northern blot analysis. Some parameters about the dual transgenic mice indicated that there were fewer litters than that of normal mice. The ratio of du     

Molecular cloning of mouse PSP mRNA.

PSP is the most abundant translation product of mouse parotid glands where its production is co-ordinated with that of salivary amylase. The synthesis of these two proteins apparently is restricted to this tissue. In order to enable us to study common regulatory elements in the genes of the two proteins, double stranded cDNA, synthesized for parotid gland poly (A)+ RNA, was cloned. DNA sequencing of three clones complementary to the most abundant messenger indicated overlap and resulted in a total sequence of 867 nucleotides. Translation of this sequence revealed that at one end the amino acid sequence was the same as the N-terminal sequence of PSP. The sequence contains 60 nucleotides coding for part of or the complete signal peptide, 645 nucleotides coding for the PSP protein, and 162 nucleotides that apparently are not translated. Southern blot analysis suggests a simple structure for the PSP gene in mouse and man.     

Parotid secretory protein is an HDL-associated protein with anticandidal activity

High-density lipoprotein (HDL) is part of innate immunity, protecting against infection and inflammation. Using a proteomic approach, we identified an amino acid sequence in a hamster HDL protein that showed homology to rat and mouse parotid secretory protein (PSP), a salivary protein secreted from the parotid glands. We cloned the cDNA encoding a putative hamster homolog of rat and mouse PSP. Searches for conserved domains of the protein showed that the COOH terminus of hamster PSP contains a region homologous to the NH2 termini of a family of HDL-associated proteins, including LPS-binding protein, cholesteryl ester transfer protein, and phospholipid transfer protein. In mice, PSP was also associated with HDL but was not detected in very-low-density lipoprotein, low-density lipoprotein, or lipoprotein-deficient sera. In addition to salivary glands, we found that PSP mRNA was expressed in lung, testis, and ovary. The level of PSP in HDL was increased after endotoxin injection in hamsters, but not in mice. Recombinant PSP inhibits growth of Candida albicans in culture. In summary, our results showed that PSP is a novel anticandidal protein associated with HDL.     

Electron Microscopic Localization of LacZ Expression in the Proximal Convoluted Tubular Cells of the Kidney in Transgenic Mice Carrying Chimeric Erythropoietin/lacZ Gene Constructs

Regulated expression of the erythropoietin (EPO) gene in the adult kidney plays a key role in the regulation of erythropoiesis. However, uncertainty exists regarding the type of kidney cells involved in EPO gene expression. We previously showed by light microscopy that the lacZ reporter gene is expressed and inducible by hypoxia/anemia in the proximal convoluted tubular (PCT) cells of the kidneys of transgenic mice carrying the 5′-lacZ construct, in which the lacZ gene was placed downstream of a 7.0-kb mouse EPO gene segment containing 6.5 kb of the 5′-flanking sequence. We, report here the light and transmission electron microscopic examination of lacZ expression in the kidneys of transgenic mice carrying the 5′-lacZ construct and two additional constructs carrying the 6.5-kb 5′-flanking sequence with the body of the gene alone, or along with the 1.2-kb 3′-flanking sequence. The electron microscopic analyses unequivocally demonstrated that lacZ under the regulatory control of the 6.5-kb 5′-flanking sequence with or without the body of the gene and the 1.2-kb 3′-flanking sequence was expressed predominantly in the proximal convoluted tubular cells of the kidney following hypoxia induction.     

Antizyme Inhibitor 2 Hypomorphic Mice. New Patterns of Expression in Pancreas and Adrenal Glands Suggest a Role in Secretory Processes

The intracellular levels of polyamines, polycations implicated in proliferation, differentiation and cell survival, are regulated by controlling their biosynthesis, catabolism and transport. Antizymes and antizyme inhibitors are key regulatory proteins of polyamine levels by affecting ornithine decarboxylase, the rate-limiting biosynthetic enzyme, and polyamine uptake. We recently described the molecular function of a novel antizyme inhibitor (AZIN2). However, the physiological function of AZIN2 in mammals is mostly unknown. To gain insight on the tissue expression profile of AZIN2 and to find its possible physiological role, we have generated, transgenic mice with severe Azin2 hypomorphism. This mouse model expresses transgenic bacterial β-D-galactosidase as a reporter gene, under the control of the Azin2 endogenous promoter, what allows a very sensitive and specific detection of the expression of the gene in the different tissues of transgenic mice. The biochemical and histochemical analyses of β-D-galactosidase together with the quantification of Azin2 mRNA levels, corroborated that AZIN2 is mainly expressed in testis and brain, and showed for the first time that AZIN2 is also expressed in the adrenal glands and pancreas. In these tissues, AZIN2 was not expressed in all type of cells, but rather in specific type of cells. Thus, AZIN2 was mainly found in the haploid germinal cells of the testis and in different brain regions such as hippocampus and cerebellum, particularly in specific type of neurons. In the adrenal glands and pancreas, the expression was restricted to the adrenal medulla and to the Langerhans islets, respectively. Interestingly, plasma insulin levels were significantly reduced in the transgenic mice. These results support the idea that AZIN2 may have a role in the modulation of reproductory and secretory functions and that this mouse model might be an interesting tool for the progress of our understanding on the role of AZIN2 and polyamines in specific mammalian cells.     

Expression and Secretion of Human Proinsulin-B10 from Mouse Salivary Glands: Implications for the Treatment of Type I Diabetes Mellitus

Adenovirus (Ad) mediated expression of therapeutic proteins from salivary glands can result in the delivery of biologically active proteins into the circulation where they impart their physiological function. In recent years, Ad vector delivery to salivary glands (SGs) has emerged as a viable option for gene therapy. Here, we engineered a variant of human proinsulin (hProinsulin-B10) into an Ad vector and demonstrated its ability to transduce cell lines, and express a bioactive protein that induces the phosphorylation of AKT, a key insulin signaling molecule. We also examined its expression in mice following delivery of the vector to the parotid gland (PTG), the submandibular gland (SMG) or to the liver via the tail vein and assessed transgenic protein expression and vector containment for each delivery method. In all cases, hProinsulin-B10 was expressed and secreted into the circulation. Lower levels of circulating hProinsulin-B10 were obtained from the PTG while higher levels were obtained from the tail vein and the SMG; however, vector particle containment was best when delivered to the SMG. Expression of hProinsulin-B10 in the SMG of chemically induced diabetic mice prevented excessive hyperglycemia observed in untreated mice. These results demonstrate that hProinsulin-B10 can be expressed and secreted into the circulation from SGs and can function physiologically in vivo. The ability to remediate a diabetic phenotype in a model of type 1 diabetes mellitus is the first step in an effort that may lead to a possible therapy for diabetes.     

Ectopic expression of β-lactoglobulin/human serum albumin fusion genes in transgenic mice: hormonal regulation andin situ localization

We produced transgenic mice carrying the native sheep -lactoglobulin (BLG) or fusion genes composed of the BLG promoter and human serum albumin (HSA) minigenes. BLG was expressed exclusively in the mammary glands of the virgin and lactating transgenic mice evaluated. In contrast, transgenic females carrying the BLG/HSA fusion constructs also expressed the HSA RNA ectopically in skeletal muscle, kidney, brain, spleen, salivary gland and skin. Ectopic expression of HSA RNA was detected only in strains that express the transgene in the mammary gland. There was no obvious correlation between the level of the HSA RNA expressed in the mammary gland and that found ectopically. In three transgenic strains analysed, the expression of HSA RNA in kidney and skeletal muscle increased during pregnancy and lactation, whereas in the brain HSA expression decreased during lactation in one of the strains. HSA protein was synthesized in skeletal muscle and skin of strain #23 and its level was higher in lactating mice compared with virgin mice. Expression of HSA was also analysed in males and was found to be more stringently controlled than in females of the same strains.In situ hybridization analyses localized the expressed transgene in the skin, kidney, brain and salivary glands of various transgenic strains. Distinct strain-specific and cell-type specific HSA expression patterns were observed in the skin. This is in contrast to the exclusive expression of the HSA transgene in epithelial cells surrounding the alveoli of the mammary gland. Taken together, these results suggest that the absence of sufficient mammary-specific regulatory elements in the BLG promoter sequences and/or the juxtaposition of the BLG promoter with the HSA coding sequences leads to novel tissue- and cell-specific expression in ectopic tissues of transgenic mice.