HSA Gene Research Articles

The Targeting Study of Human Serum Albumin Gene

To generate transgenic porcine which expresses human serum albumin (HAS), the HSA gene targeting vector was constructed with HSA cDNA as the gene of interest and partial porcine serum albumin (PSA) gene as homologous arms which were 7.2 kb 5′ regulation sequence and 2.8 kb genomic sequence from the first intron to the fourth intron, respectively. The resistant gene neo was inserted into intron 1 and tk was ligated to the 3′ end of the construct. Linearized targeting construct DNA was introduced into the fibroblast cells obtained from porcine fetus by electroporation. The positive-negative selection was performed and survival clones were screened by PCR and Southern blot. Three colonies with correct homologous recombination were obtained. Our results set a good basis for the establishment of transgenic porcine by gene target and nuclear transfer methods.

Albumin locust valley: A new case of analbuminemia

Objective: To discover and document cases of the rare disease analbuminemia. Design and methods: Blood specimens of a subject were analyzed by routine clinical laboratory procedures, by immunochemical tests for albumin, and by electrophoresis with immunofixation using anti-human serum albumin (HSA). Single-strand conformational polymorphism (SSCP), heteroduplex analysis (HA), and DNA sequencing of the 14 exons of the HSA gene were conducted on DNA from leukocytes. Results: Albumin concentration was 0.003 g/L; serum globulins and cholesterol were elevated. Immunoelectrophoresis showed no trace of albumin in any of the serum components. The coding region plus all mRNA splice sites were normal and mutation-free, and SSCP and HA showed no abnormalities. Conclusions: Data define a bona fide case of analbuminemia. We suggest that the mutation causing the analbuminemic trait in this subject might involve a remote regulatory element.

Congenital Analbuminemia attributable to Compound Heterozygosity for Novel Mutations in the Albumin Gene

Congenital analbuminemia is a rare autosomal recessive disorder characterized by the absence or very low concentrations of serum albumin (HSA) (1). The disorder is conventionally defined as an HSA <1 g/L (as determined by immunoassay) associated with normal liver function and absence of protein loss (2). The incidence of congenital analbuminemia is estimated to be <1 in 1 million births, without sex or race predilection. To date, 39 cases of congenital analbuminemia have been reported, 16 of which were diagnosed in children (3). Despite multiple functions of HSA (4), its absence is a relatively tolerable condition. Except for associated hyperlipidemia, minor edema, and mild fatigability, analbuminemic individuals suffer few adverse symptoms (3)(5)(6). Such relative mildness of symptoms is attributed to a compensatory increase in hepatic biosynthesis of other plasma proteins (7)(8). Congenital analbuminemia is attributable to defects in the gene coding for HSA. The HSA gene is located on chromosome 4 and is split into 15 exons by 14 intervening introns (9). The identification of multiple mutations that cause analbuminemia clearly indicates that it is a genetically heterogeneous disorder. Six of these mutations introduced stop codons (10)(11)(12)(13), and 2 caused splicing defects and determined a premature termination of the translation (14)(15). All were found to be present in the homozygous state. In the present study we characterized another case of congenital analbuminemia caused by 2 newly identified mutations in an Italian family. The proband was a 29-year-old Italian man with an HSA of 10–12 mg/L as determined by immunoassay. His medical history has been reported elsewhere (16)(17). The proband’s relatives had HSA concentrations in the lower end of the reference interval (father, 35 g/L; mother, 33 g/L; sister, 42 g/L). All gave informed …

A Novel GPIbα Binding Protein on Streptococcus Gordonii Induces Platelet Rolling at Low Shear.

Infective endocarditis is associated with considerable morbidity and mortality and oral Streptococci are the priniciple causative organism. Platelet adhesion and subsequent aggregation play a critical role in the pathogenesis and dissemination of the infective process. The mechanism by which S. gordonii interacts with and subsequently leads to platelet activation is currently unknown. Previously we described the ability of Streptococcus sanguis to induce platelet aggregation via the platelet vonWillebrand factor receptor, GPIbα, however identification of the bacterial protein involved was not established. Here we describe the novel interaction between a Streptococcal protein that triggers aggregation via platelet GPIbα, in a unique fashion.Wildtype S. sanguis (133–79) and S. gordonii (DL1) support platelet adhesion and induce αIIbβ3 dependent platelet aggregation. Pre-incubation of platelets with an anti-GPIbα antibody inhibited the adhesion (61±6%, p<0.001) and abolished platelet aggregation (0% of control, p<0.001) induced by S. gordonii. These results suggest that GPIbα is a key receptor for recognition of platelets by streptococci. Passing a mutanolysin cell wall extract of S. sanguis through a GPIb affinity column identified a highly glycosylated protein, Hsa, also present on S. gordonii. S. gordonii DL-1 deficient in the Hsa gene, was generated by allelic exchange with an antibiotic resistance cassette. Platelet adhesion to S. gordonii ΔHsa was reduced by 41±5%, (p<0.001), however aggregation was unaffected (49±7% vs 55±5% wt). To confirm that Hsa was binding to platelet GPIbα we immobilised soluble GPIbα (glycocalicin, 0.2μg/ml) on a 96 well plate. Wildtype S. gordonii bound to immobilised glycocalicin, however, the Hsa mutant adhered significantly less (9±3% of wildtype, p<0.001). Furthermore, wildtype S. gordonii induced αIIbβ3 dependent platelet aggregation in washed platelets in the absence of vWf, suggesting a direct interaction between Hsa and GPIbα. We further investigated the interaction between GPIbα and Hsa under shear. Experiments were recorded in real time for a period of 8 minutes. Upon commencement of shear (50s-1), platelets interacted with S. gordonii DL-1 with a rolling fashion followed by firm adhesion. This adhesion was completely inhibited by addition of an anti-GPIbα antibody. When platelets were sheared over immobilized S. gordonii ΔHsa no interaction was observed. In addition, S. gordonii DL-1 failed to interact with platelets at the higher shear rate of 500s-1.Collectively, these results identify a unique interaction between S. gordonii Hsa and platelet GPIbα. This interaction occurs at static and low shear but not at high shear, which is in direct contrast to the interaction between vwf and GPIbα. Furthermore, we propose that the platelet interaction with S. gordonii appears to be a 2 step process. Firstly, platelets roll across the S. gordonii via a GPIbα - Hsa interaction. Following this a second more stable interaction firmly immobilizes the platelet, thereby facilitating the intravascular colonization of a septic plaque.

The ALEU2 gene--a new component for an Arxula adeninivorans-based expression platform.

The ALEU2 gene, encoding beta-isopropylmalate dehydrogenase, was isolated from the non-conventional yeast Arxula adeninivorans. The isolated gene harbours an open reading frame of 1086 bp, encoding a putative protein of 362 amino acids. The derived protein sequence shares a high degree of homology with other fungal beta-isopropylmalate dehydrogenases thus confirming the identity of the gene. The isolated ALEU2 gene was tested for its suitability to complement the auxotrophy of an A. adeninivorans aleu2 host. For this purpose the plasmid pAL-ALEU2m which contains the ALEU2 gene as a selection marker and the 25S rDNA for targeting was employed in transformation experiments. Transformants harboured a single copy of the heterologous DNA and were found to be mitotically stable. For assessment of heterologous gene expression, two model genes were incorporated into the vector: the GFP gene, encoding intracellular green fluorescent protein, and the HSA gene, encoding the secreted human serum albumin. For expression control, both gene sequences were fused to the constitutive A. adeninivorans-derived TEF1 promoter and the Saccharomyces cerevisiae-derived PHO5 terminator. In the respective recombinant strains the GFP was localised in the cytoplasm, whereas more than 95% of the HSA accumulated in the culture medium. In initial fermentation trials using a 200-ml shake flask, maximal HSA product levels were observed after 96 h of cultivation.

High-level production and secretion of recombinant proteins by the dimorphic yeastArxula adeninivorans

The non-conventional dimorphic thermo- and salt-resistant yeast Arxula adeninivorans has been developed as a host for heterologous gene expression. For assessment of the system two model genes have been selected: the GFP gene encoding the intracellular green fluorescent protein, and the HSA gene encoding the secreted human serum albumin. The expression system includes two host strains, namely A. adeninivorans LS3, which forms budding cells at 30°C and mycelia at >42°C, and the strain A. adeninivorans 135, which forms mycelia at temperatures as low as 30°C. For expression control the constitutive A. adeninivorans-derived TEF1-promoter and S. cerevisiae-derived PHO5-terminator were selected. The basic A. adeninivorans transformation/expression vector pAL-HPH1 is further equipped with the Escherichia coli-derived hph gene conferring hygromycin B resistance and the 25S rDNA from A. adeninivorans for rDNA targeting. Transformants were obtained for both budding cells and mycelia. In both cell types similar expression levels were achieved and the GFP was localised in the cytoplasm while more than 95% of the HSA accumulated in the culture medium. In initial fermentation trials on a 200-ml shake flask scale maximal HSA product levels were observed after 96 h of cultivation.

High-level production and secretion of recombinant proteins by the dimorphic yeast Arxula adeninivorans.

The non-conventional dimorphic thermo- and salt-resistant yeast Arxula adeninivorans has been developed as a host for heterologous gene expression. For assessment of the system two model genes have been selected: the GFP gene encoding the intracellular green fluorescent protein, and the HSA gene encoding the secreted human serum albumin. The expression system includes two host strains, namely A. adeninivorans LS3, which forms budding cells at 30 degrees C and mycelia at >42 degrees C, and the strain A. adeninivorans 135, which forms mycelia at temperatures as low as 30 degrees C. For expression control the constitutive A. adeninivorans-derived TEF1-promoter and S. cerevisiae-derived PHO5-terminator were selected. The basic A. adeninivorans transformation/expression vector pAL-HPH1 is further equipped with the Escherichia coli-derived hph gene conferring hygromycin B resistance and the 25S rDNA from A. adeninivorans for rDNA targeting. Transformants were obtained for both budding cells and mycelia. In both cell types similar expression levels were achieved and the GFP was localised in the cytoplasm while more than 95% of the HSA accumulated in the culture medium. In initial fermentation trials on a 200-ml shake flask scale maximal HSA product levels were observed after 96 h of cultivation.

Establishment and initial characterization of the ovine mammary epithelial cell line NISH.

Analysis of the molecular mechanisms involved in the differentiation and formation of the characteristic three-dimensional structures of the developing mammary gland of the major milk-producing livestock (ducts, end buds, and alveoli) requires in vitro model cell cultures. The few cell lines that have been established from dairy animals do not fully reproduce the entire program of mammary differentiation. Here we present the initial characterization of a unique mammary epithelial cell line derived spontaneously from midpregnant sheep (NISH). These cells form in vitro functional structures resembling ducts, lateral buds, and alveoli that secrete beta-lactoglobulin (BLG) in an ECM (extracellular matrix)-dependent manner. Interestingly, the presence of growth hormone dramatically increased BLG secretion from NISH cells cultured on ECM. It appears that GH is required not only to establish the structural organization but also is continuously needed to maintain BLG expression. Stable transfection of NISH cells with BLG/Human Serum Albumin (HSA) hybrid gene constructs revealed that the relative level of expression was comparable to the in vivo secretion of HSA in transgenic mice carrying these gene sequences. No expression could be detected in cells transfected with hybrid genes carrying either HSA cDNA or the entire HSA gene, and HSA expression was dependent on the presence of intronic sequences. These results demonstrate that NISH cells may prove a useful tool for studying the differentiation and organogenesis of mammary epithelial cells under defined culture conditions. Furthermore, transfected NISH cells may be an alternative for the transgenic mouse model in evaluating the potential of gene constructs to be efficiently expressed in the mammary gland of transgenic farm animals.

Isolation and analysis of different subpopulations of normal human breast epithelial cells after their infection with a retroviral vector encoding a cell surface marker.

The use of gene transfer procedures has greatly facilitated the investigation of cell lineage relationships and other developmental processes in a variety of primary tissues. In this report we described the infection and selection of primary human breast epithelial cells using retroviral vectors (Jzen-HSA-NEO and MSCV-HSA.NEO) containing the complete 228 bp coding sequence of a murine cell surface marker (Heat Stable Antigen, HSA) as well as the neomycin resistance (neo(r)) gene. Expression of the transduced HSA gene was detectable using either flow cytometry or immunohistochemistry after staining cells with an anti-murine HSA-specific antibody (M1/69). Expression of the transduced neo(r) gene conferred resistance to G418. In initial experiments with the MCF-7 breast cancer cell line, continued expression of both markers was demonstrated in a high proportion of cells for at least 4 weeks after their infection by positive M1/69 staining of cells that had been selected by prior incubation in G418. Evidence of gene transfer to early stage (< 9 days old) primary cultures of normal human breast epithelial cells (15 experiments with cells from 12 normal individuals) was also obtained using an infection protocol in which these calls were exposed to helper-free viral supernatants (2 incubations, 4 to 6 hr each) after being subcultured for 12 to 18 hr to increase their rate of proliferation. The presence of 5-50% (mean = 26%) HSA+ cells was demonstrated in these experiments within 5 days after their infection and the HSA+ populations included both myoepithelial and luminal phenotypes. The transduced (HSA+) cells within both of these subpopulations could also be separately isolated by FACS and subcultured. These results should provide an important starting point for future studies of genetically modified or marked primary human breast epithelial cell populations.

Altered Erythrocytes and a Leaky Block in B-Cell Development in CD24/HSA-Deficient Mice

AbstractThe heat stable antigen (HSA, or murine CD24) is a glycosyl phosphatidylinositol-linked surface glycoprotein expressed on immature cells of most, if not all, major hematopoietic lineages, as well as in developing neural and epithelial cells. It has been widely used to stage the maturation of B and T lymphocytes because it is strongly induced and then repressed again during their maturation. Terminally differentiated lymphocytes, as well as most myeloid lineages, are negative for HSA. Erythrocytes are an exception in that they maintain high levels of HSA expression. HSA on naive B cells has been shown to mediate cell-cell adhesion, while HSA on antigen-presenting cells has been shown to mediate a costimulatory signal important for activating T lymphocytes during an immune response. Here, we characterize mice that lack a functional HSA gene, constructed by homologous recombination in embryonic stem cells. While T-cell and myeloid development appears normal, these mice show a leaky block in B-cell development with a reduction in late pre-B and immature B-cell populations in the bone marrow. Nevertheless, peripheral B-cell numbers are normal and no impairment of immune function could be detected in these mice in a variety of immunization and infection models. We also observed that erythrocytes are altered in HSA-deficient mice. They show a higher tendency to aggregate and are more susceptible to hypotonic lysis in vitro. In vivo, the mean half-life of HSA-deficient erythrocytes was reduced. When infected with the malarial parasite Plasmodium chabaudi chabaudi, the levels of parasite-bearing erythrocytes in HSA-deficient mice were also significantly elevated, but the mice were able to clear the infection with kinetics similar to wild-type mice and were immune to a second challenge. Thus, apart from alterations in erythrocytes and a mild block in B-cell development, the regulated expression of HSA appears to be dispensable for the maturation and functioning of those cell lineages that normally express it.

The heat stable antigen (mouse CD24) gene is differentially regulated but has a housekeeping promoter.

Expression of the mouse heat stable antigen (HSA or mouse CD24) shows tissue-specific as well as developmental regulation. During the maturation of several hematopoietic lineages, HSA expression is generally high in immature precursor cells and low or absent in terminally differentiated cells. We present evidence suggesting that this regulation of the HSA gene (Cd24a) occurs at the transcriptional level. In addition, sequence and methylation analysis of the Cd24a promoter revealed characteristics of both "housekeeping" and tissue-specific promoters, including a methylation-free, HpaII tiny fragment (HTF) island, multiple putative SP1 and AP-2 consensus binding sites, and a TATA box. Functional analysis of a 0.6-kilobase DNA fragment containing these elements fused to the CAT reporter gene in transient transfection experiments showed activity in both HSA expressing and non-expressing cell lines with a strength similar to that of the herpes-simplex virus-thymidine kinase promoter. Large fragments from the flanking region of the Cd24a promoter did not influence the ubiquitous nature of this promoter. Finally, we mapped the Cd24a, Cd24b, and Cd24c genes to mouse chromosomes 10, 8, and 14 respectively.

Efficient generation of functional transgenes by homologous recombination in murine zygotes.

To assess the feasibility of generating functional transgenes directly via homologous recombination between microinjected DNA fragments, three overlapping genomic DNA fragments, together constituting the human serum albumin (hSA) gene, were coinjected into murine zygotes. The resulting transgenic mice were analyzed for structure and expression of the transgene. All transgenic mice carried recombined hSA DNA fragments and 74% contained a reconstituted hSA gene. HSA expression could be detected in liver and serum in most (72%) of these animals. Only correctly sized hSA transcripts were observed. Transgenic hSA could not be distinguished from the human serum-derived protein by radioimmunoassay or Western blotting. The high frequency and accuracy of homologous recombination in murine zygotes reported here allows the efficient generation of relatively large transgenes.

Stable multicopy vectors for high-level secretion of recombinant human serum albumin by Kluyveromyces yeasts.

We have designed stable pKD1 derivatives for efficient secretion of recombinant human serum albumin (rHSA) by industrial strains of Kluyveromyces yeasts. A comparison of this multi-copy expression system with isogenic cassettes integrated at chromosomal loci demonstrated that high level secretion of rHSA is a function of gene dosage in K. lactis. Various signal sequences could be used, and the secretion levels were independent of the presence of the native pro peptide. The mitotic stability of the pKD1-based expression vectors was found to be species and strain dependent and was influenced by promoter strength and culture conditions. Vector stability was drastically enhanced when the HSA gene was expressed from an inducible promoter: 90% of the transformed cells still harbored the vector after 100 generations of non-selective growth in uninduced culture conditions. Secretion levels in the range of several grams per liter of correctly folded and processed rHSA were obtained at the pilot scale, thus making the industrial production of pharmaceutical-grade, Kluyveromyces-derived rHSA economically feasible.