Murine Erythropoietin Gene: Cloning, Expression, and Human Gene Homology

Shoemaker, Charles B.; Mitsock, L D

doi:10.1128/mcb.6.3.849-858.1986

Cited by 6 publications

(1 citation statement)

References 38 publications

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“…If a comparison is made between human and murine EPO at the DNA or protein level, a homology of about 80% is found (Shoemaker & Mitsock, 1986; McDonald et al , 1986). Despite this, the polyclonal anti‐human EPO antibodies in our human EPO ELISA recognized the murine EPO molecule to a far lesser extent than the human.…”

Section: Discussionmentioning

confidence: 99%

An ELISA specific for murine erythropoietin

et al. 1999

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Summary. Murine recombinant erythropoietin (EPO) was purified from an EPO-producing cell line and used for the production of polyclonal monospecific anti-murine EPO antibodies in rabbits. The anti-mouse EPO antibodies were purified by two affinity chromatography procedures. In order to obtain the most sensitive ELISA, different antibody combinations were tested in the ELISA sandwich assay. The best combination was achieved with an anti-human EPO antibody as coating and the biotinylated anti-murine EPO antibody as detecting antibody. With this sandwich-ELISA a sensitive standard curve in the range of 0·6-30 mU/ ml could be established. The assay provides a sensitive and reliable measure of murine EPO in serum and cell culture supernatants ranging from normal to highly elevated EPO levels.Keywords: murine erythropoietin, purification, polyclonal antibodies, sandwich-ELISA, specificity.Erythropoietin (EPO) is a glycoprotein growth factor responsible for the proliferation and differentiation of early and late erythropoietic progenitor cells. When recombinant human EPO became available, several immunological assays for measuring EPO in human serum were established (Jelkmann, 1992). In our own laboratory we developed a sensitive and reliable sandwich ELISA for human EPO (Noé et al, 1992). However, this human ELISA was not sufficiently sensitive to efficiently measure mouse EPO in serum or culture supernatants from murine-derived cells. In this report we describe a sandwich ELISA specific for mouse EPO, using polyclonal antibodies produced against murine recombinant EPO. MATERIALS AND METHODS Purification of murine EPO (m-EPO).The source for the purification of m-EPO was the cell line Ne-3, kindly provided by Dr W. Ostertag (Heinrich-Pette-Institut, Universität Hamburg, Germany). This mouse fibroblast cell line contained the entire coding sequence of the mouse EPO gene. The cells were first cultivated in CG medium (Vitromex, Vilshofen, Germany) with 10% fetal bovine serum (FBS, Boehringer Mannheim, Germany), the concentration of which was gradually reduced in order to obtain serum-free conditions. After cultivation in CG medium in the absence of FBS for 14 d, the cells were centrifuged and the supernatant was mixed with 1/20 of the volume of 20 × phosphatebuffered saline (PBS), containing 2% Tween 80, 2% sodium azide (NaN 3 ) and stored at ¹20ЊC until processed.The following three chromatographic steps were performed using a FPLC (Pharmacia, Freiburg, Germany). The first step was chromatography of the supernatant over an agarose wheat germ lectin (Pharmacia) column equilibrated with PBS, containing 0·1% NaN 3 , 0·1% Tween 80. The column was washed with the equilibration buffer and the bound protein was eluted with 0·3 M N-acetylglucosamine (Serva, Heidelberg, Germany) in equilibration buffer. The eluted fractions were measured at 280 nm. In addition, EPO present in the eluted fractions was also measured with an EPO ELISA (Noé et al, 1992) modified for recombinant m-EPO (Boehringer Mannheim, Germany). The fractions wit...

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Section: Discussionmentioning

confidence: 99%

An ELISA specific for murine erythropoietin

et al. 1999

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Hypoxic stress tolerance of the blind subterranean mole rat: Expression of erythropoietin and hypoxia-inducible factor 1α

Shams

Avivi

Nevo

2004

Proc. Natl. Acad. Sci. U.S.A.

130

108

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Blind subterranean mole rats (Spalax, Spalacidae) evolved adaptive strategies to cope with hypoxia that climaxes during winter floods in their burrows. By using real-time PCR, we compared gene expression of erythropoietin (Epo), a key regulator of circulating erythrocytes, and hypoxia-inducible factor 1␣ (HIF-1␣), Epo expression inducer, in the kidneys of Spalax and white rats, Rattus norvegicus. Our results show significantly higher, quicker, and longer responses to different O 2 levels in Spalax compared with Rattus. (i) In normoxia, both Spalax and Rattus kidneys produce small amounts of Epo. Maximal expression of Rattus Epo is noticed after a 4-h hypoxia at 6% O 2. Under these conditions, Spalax Epo levels are 3-fold higher than in Rattus. After 24 h of 10% O 2, Spalax Epo reaches its maximal expression, remarkably 6-fold higher than the maximum in Rattus; (ii) the HIF-1␣ level in normoxia is 2-fold higher in Spalax than in Rattus. Spalax HIF-1␣ achieves maximal expression after 4-h hypoxia at 3% O 2, a 2-fold increase compared with normoxia, whereas no significant change was detected in Rattus HIF-1␣ at any of the conditions studied; (iii) at 6% O2 for 10 h, in which Rattus cannot survive, Epo and HIF-1␣ levels in Spalax galili, living in heavily flooded soils, are higher than in Spalax judaei, residing in light aerated soil. We suggest that this pattern of Epo and HIF-1␣ expression is a substantial contribution to the adaptive strategy of hypoxia tolerance in Spalax, evolved during 40 million years of evolution to cope with underground hypoxic stress.

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Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation

Rizzuto

Cappelletti

Maione

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

308

228

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We show that an electric treatment in the form of high-frequency, low-voltage electric pulses can increase more than 100-fold the production and secretion of a recombinant protein from mouse skeletal muscle. Therapeutical erythopoietin (EPO) levels were achieved in mice with a single injection of as little as 1 g of plasmid DNA, and the increase in hematocrit after EPO production was stable and long-lasting. Pharmacological regulation through a tetracycline-inducible promoter allowed regulation of serum EPO and hematocrit levels. Tissue damage after stimulation was transient. The method described thus provides a potentially safe and low-cost treatment for serum protein deficiencies.Genes can be transferred into skeletal muscle cells of rodents and primates by intramuscular injection of plasmid DNA, and the resulting gene expression has been reported to last as long as several months (1, 2). Similarly, various viral vectors such as adenoviral, retroviral, and AAV-based vectors (3), have been used to transduce myofibers in vivo. The i.m. injection of plasmid DNA, however, has several advantages over viral vectors. First, plasmid DNA vectors are easier to construct and can be prepared as pharmaceutical-grade solutions (4) without the risk of contamination with wild-type infectious particles. Second, previous infection by wild-type adenovirus or AAV may induce a neutralizing antibody response that could preclude administration of the recombinant virus. In contrast, anti-DNA antibodies have never been detected in experiments of muscle DNA injection (2), therefore it is possible to readminister plasmid DNA by i.m. injection if repeated therapy or escalation is required.Despite the promise of i.m. injection of plasmid vectors for treating serum protein deficiencies, several important issues remain to be addressed before this approach becomes feasible for human gene therapy. The potential clinical usefulness of direct gene transfer to muscle of plasmid DNA is in fact limited by the low and highly variable level of gene expression (1, 2, 5, 6). Therefore, although DNA injection is potentially very powerful as a vaccination method because a low level of gene expression is sufficient to trigger immunoresponses, it is necessary to increase the efficiency of DNA uptake after i.m. injection of plasmid vectors before using this technique as a standard gene correction procedure.One of the most efficient methods implemented to achieve gene transfer and expression in mammalian cells is based on electric pulses (7). Electroporation has been used to introduce foreign DNA in different cell types (7), but it has also recently met with some success in in vivo applications. Gene transfer by electrical permeabilization has been obtained in skin (8, 9), corneal endothelium (10), melanoma (11), brain (12), liver, (13) and muscle (14) of experimental animals.We have shown previously that electropermeabilization can increase severalfold the uptake by rat muscle of a plasmid encoding the Escherichia coli lacZ gene (15). In this study...

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Murine Erythropoietin Gene: Cloning, Expression, and Human Gene Homology

Cited by 6 publications

References 38 publications

An ELISA specific for murine erythropoietin

An ELISA specific for murine erythropoietin

Hypoxic stress tolerance of the blind subterranean mole rat: Expression of erythropoietin and hypoxia-inducible factor 1α

Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation

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