Expression of human erythropoietin cDNA in human lymphoblastoid Namalwa cells: the inconsistency of a stable expression level with transient expression efficiency

doi:10.1016/0378-1119(89)90004-8

Gene

Volume 76, Issue 1, 15 March 1989, Pages 19-26

https://doi.org/10.1016/0378-1119(89)90004-8 Get rights and content

Abstract

Recombinant plasmids for the expression of human erythropoietin (EPO) cDNA in Namalwa cells were constructed. From the results of the EPO expression efficiency in transiently transfected cells, it was found that the simian virus 40 (SV40) early promoter directs EPO synthesis more efficiently in Namalwa cells than does the long terminal repeat promoter of Rous sarcoma virus and that the 3′-noncoding sequence including splice junction and polyadenylation site derived from the rabbit β-globin gene are more effective than those of the SV40 early gene. However, in stable transformants, no simple relationship was found between the expression level of EPO cDNA and the structure of the introduced expression vectors.

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Cited by (6)

Rapid determination of gap junction formation using HeLa cells microinjected with cDNAs encoding wild-type and chimeric connexins
1998, Biochemical and Biophysical Research Communications
A procedure for rapidly determining the functionality of gap junctions constructed of recombinant connexins in communication-deficient HeLa cells is described. Nuclear microinjection of cDNA encoding wild-type connexins (Cx) 26, 32, 43, and a range of connexin-aequorin (Cx-Aeq) chimerase resulted in generation of gap junction intercellular communication channels. Expression of recombinant protein was detected in >95% of cells 18–72 h following nuclear microinjection, and the functionality of the channels generated was determined according to their ability to transfer the fluorescent dye tracers Lucifer yellow and propidium iodide. The dye transfer results obtained correlated closely with other published studies using stably transfected cells and yet are obtained as rapidly as 18 h following microinjection of cDNA. Expression of a truncated form of Cx43 (Cx43^Δ244) by this new method indicated diminished intercellular transfer of both dyes and supports a channel-gating mechanism that postulates interaction between the carboxyl tail and the intracellular loop.
Efficient production of biologically active human recombinant proteins in human lymphoblastoid cells from integrative and episomal expression vectors
1994, Gene
The ability of human lymphoblastoid cells to secrete large amounts of biologically active human hematopoietic growth factors from adenovirus-based expression vectors was investigated. The gene for human erythropoietin (EPO) was inserted into integrative (pTS39) and episomal (pTS53) vectors. Cell clones, originating from pTS39 or pTS53-transfected and stably selected cells, secreted recombinant human EPO (re-hEPO) at similar levels. The highest production, 60 u/10⁶ cells per 24 h, was obtained from a subclone of pTS39-transfected cells, grown in nonselective medium. The re-hEPO was shown to be biologically active in vivo by incorporation of ⁵⁹Fe into red blood cells of polycythemic mice and in vitro by the proliferative response of the EPO-dependent cell line UT7. The purified protein of 36 kDa in SDS-PAGE slightly differed from re-hEPO from CHO cells. pTS39 vector was integrated at 15–30 copies per genome, whereas the pTS53 vector replicated at 10 copies per cell. Genes encoding human interleukin-3 (IL-3) and granulocytemacrophage colony-stimulating factor (GM-CSF) were also expressed in the integrative system as biologically active growth factors, demonstrating that our host-vector system allows the expression of any little gene or cDNA and efficient secretion of the re-protein produced.
Structures of Mucin-Type Sugar Chains on Human Erythropoietins Purified from Urine and the Culture Medium of Recombinant Chinese Hamster Ovary Cells
1993, Archives of Biochemistry and Biophysics
Less is known about the mucin-type sugar chains attached to human erythropoietin as compared with N-linked sugar chains which structures and function have been well studied. In this study, we purified urinary human erythropoietin from three independent groups of aplastic anemia patients, and analyzed the structures of mucin-type sugar chains as well as that obtained from recombinant human erythropoietin produced by Chinese hamster ovary cells. Unlike the N-linked sugar chains, the mucin-type sugar chains are totally different between the urinary and the recombinant erythropoietins. All of the three independent samples of urinary erythropoietin contained oligosaccharides with the structures of ±Neu5Acα2 → 6GalNAc, while recombinant human erythropoietin contained those with the structures of Neu5Acα2 → 3Galβ1 → 3(Neu5Acα2 → 6)Ga1NAc, Neu5Acα2 → 3Galβ1 → 3GalNAc, and Galβ1 → 3(Neu5Acα2 → 6)GalNAc.
Stable expression of human tissue-type plasminogen activator regulated by β-actin promoter in three human cell lines: HeLa, WI-38 VA13 and KMS-5
1991, BBA - Gene Structure and Expression
A high-level and stable expression system of human tissue-type plasminogen activator (t-PA) was accomplished in human cells by selecting a promoter and a host cell line. First, we have constructed two types of t-PA expression plasmids containing 3 kb of the human β-actin promoter region or 0.3 kb of SV40 early promoter region and these plasmids were transfected into HeLa cells, respectively, and the resulting transfectants were found to secrete various amounts of t-PA derived from the plasmids to the culture media. Southern blot analysis revealed that the β-actin promoter was more efficient than the SV40 early promoter with regard to the expression level per single copy of the t-PA gene in the transfected HeLa cells. Next, the t-PA expression plasmid containing the β-actin promoter was also transfected into WI-38 VA13 cells, a human fibroblastic cell line, and KMS-5 cells, a human lymphoid cell line, in order to compare the expression ability of the promoter among these three cell lines. Some of the transfectants from both cell lines were also found to produce t-PA. It was also found that the expression levels in HeLa and WI-38 VA13 seemed to be more efficient than that in KMS-5.
High-level expression of human erythropoietin cDNA in stably transfected Namalwa cells
1989, Journal of Fermentation and Bioengineering
We have optimized the expression of an introduced DNA coding for human erythropoietin (EPO) in stably transfected human B lymphoblastoid Namalwa cells. The results of transient expression of genomic and cDNA for EPO under the control of the SV40 early promoter-enhancer unit suggested that cDNA yields higher expression of EPO than genomic DNA. An intervening sequence was required for efficient expression, although even an intronless transcriptional unit could still express EPO. Stable Namalwa cells that secreted 1 to 2 μg of EPO/10⁶ cells/d were then established by transfection with DNA constructs consisting of the SV40 early promoter, EPO cDNA, and the splicing junction and polyadenylation site derived from the SV40 T antigen gene or the rabbit β-globin gene. One of these transformant cell lines was easily adapted to grow in serum-free medium.
Recombinant Human Erythropoietin Produced by Namalwa Cells
1989, DNA

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Gene

Abstract

References (31)

A lymphocyte-specific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes

Cell

The expression of recombinant DNA products in mammalian cells

Trends Biotechnol.

Selective extraction of polyoma DNA from infected mouse cell cultures

J. Mol. Biol.

Amplification and expression of sequences cotransfected with a modular dihydrofolate reductase complementary DNA gene

J. Mol. Biol.

At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells

J. Mol. Biol.

Purification of human erythropoietin

J. Biol. Chem.

A comparison of bovine growth hormone expression directed by bGH genomic or intronless DNA in transiently transfected eukaryotic cells

Gene

Cytoplasmic dot hybridization

J. Biol. Chem.

The tac promoter: a functional hybrid derived from the trp and lac promoters

A quantitative bioassay for erythropoietin using mouse fetal liver cells

Exp. Hematol.

Interferon production from human cell cultures

Nonreciprocal exchanges of information between DNA duplexes coinjected into mammalian cell nuclei

Mol. Cell. Biol.

Synthesis of human fibroblast Interferon by E. coli

Nucleic Acids Res.

The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA-mediated transfection

High efficiency DNA-mediated transformation of primate cells

Science

Cited by (6)

Rapid determination of gap junction formation using HeLa cells microinjected with cDNAs encoding wild-type and chimeric connexins

Efficient production of biologically active human recombinant proteins in human lymphoblastoid cells from integrative and episomal expression vectors

Structures of Mucin-Type Sugar Chains on Human Erythropoietins Purified from Urine and the Culture Medium of Recombinant Chinese Hamster Ovary Cells

Stable expression of human tissue-type plasminogen activator regulated by β-actin promoter in three human cell lines: HeLa, WI-38 VA13 and KMS-5

High-level expression of human erythropoietin cDNA in stably transfected Namalwa cells

Recombinant Human Erythropoietin Produced by Namalwa Cells