Aberrant phenotypes of transgenic mice expressing dimeric human erythropoietin

Reproductive Biology and Endocrinology Aberrant phenotypes of transgenic mice expressing dimeric human erythropoietin Seong-Jo Yun 0 Purevjargal Naidansuren 0 Bo-Woong Sim 0 Jong-Ju Park 0 Cha-Won Park 0 Tseeleema Nanjidsuren 1 Myung-Hwa Kang 1 Sue-Yun Hwang 0 Jong-Taek Yoon 0 Kwan-Sik Min 0 0 Animal Biotechnology, Graduate School of Bio & Information Technology, Institute of Genetic Engineering, Hankyong National University , Ansung 456- 749 , Korea 1 Department of Food and Nutrition, Hoseo University , Asan 336- 795 , Korea Background: Dimeric human erythropoietin (dHuEPO) peptides are reported to exhibit significantly higher biological activity than the monomeric form of recombinant EPO. The objective of this study was to produce transgenic (tg) mice expressing dHuEPO and to investigate the characteristics of these mice. Methods: A dHuEPO-expressing vector under the control of the goat beta-casein promoter, which produced a dimer of human EPO molecules linked by a 2-amino acid peptide linker (Asp-Ile), was constructed and injected into 1-cell fertilized embryos by microinjection. Mice were screened using genomic DNA samples obtained from tail biopsies. Blood samples were obtained by heart puncture using heparinized tubes, and hematologic parameters were assessed. Using the microarray analysis tool, we analyzed differences in gene expression in the spleens of tg and control mice. Results: A high rate of spontaneous abortion or death of the offspring was observed in the recipients of dHuEPO embryos. We obtained 3 founder lines (#4, #11, and #47) of tg mice expressing the dHuEPO gene. However, only one founder line showed stable germline integration and transmission, subsequently establishing the only transgenic line (#11). We obtained 2 F1 mice and 3 F2 mice from line #11. The dHuEPO protein could not be obtained because of repeated spontaneous abortions in the tg mice. Tg mice exhibited symptoms such as short lifespan and abnormal blood composition. The red blood cell count, white blood cell count, and hematocrit levels in the tg mice were remarkably higher than those in the control mice. The spleens of the tg mice (F1 and F2 females) were 11- and -21-fold larger than those of the control mice. Microarray analysis revealed 2,672 spleenderived candidate genes; more genes were downregulated than upregulated (849/764). Reverse transcriptasepolymerase chain reaction (RT-PCR) and quantitative real-time PCR (qRT-PCR) were used for validating the results of the microarray analysis of mRNA expression. Conclusions: In conclusion, dHuEPO tg mice caused excessive erythrocytosis that led to abnormal blood composition, short lifespan, and abnormal splenomegaly. Further, we identified 2,672 genes associated with splenomegaly by microarray analysis. These results could be useful in the development of dHuEPO-producing tg animals. - Background Erythropoietin (EPO), a 30.4-kDa glycoprotein hormone secreted mainly by peritubular cells of the adult kidney, is the major factor regulating red blood cell (RBC) production [1]. Recombinant human EPO (rhEPO) has been approved for the treatment of anemia resulting from chronic renal failure, cancer chemotherapy, AIDS, etc. [2-4]. Administration of rhEPO as a potential therapeutic agent can reduce the necessity for blood transfusions and improve the patients quality of life. Although rhEPO may be beneficial for the patients, the price of such a treatment prevents its use as a long-term intravenous treatment. Therefore, various strategies have been used to stimulate erythropoiesis. Many approaches to extend the half-life of EPO through genetic changes or chemical modification of native EPO have been considered in detail [5,6]. All these strategies have shown some effect on extending the half-life and enhancing the activities of rhEPO. Particularly, dimerization of 2 rhEPO peptides can significantly enhance the biological activity of the hormone; this is because the dimer has 2 high-affinity binding sites, resulting in better binding to the EPO receptor than is observed with the monomeric form of recombinant rhEPO [7-9]. Similarly, the longer half-life of novel erythropoietin stimulating protein (NESP), which was created by the introduction of 2 extra N-linked carbohydrate addition sites into the primary sequence of EPO, is likely to afford it a clinical advantage over rhEPO by allowing less frequent dosing in patients treated for anemia [10]. An EPO chimeric protein, constructed by fusing the carboxyl-terminal peptide of a human chorionic gonadotropin-b subunit bearing 4 O-linked oligosaccharide recognition sites with the coding sequence of human EPO cDNA, did not show altered secretion, receptor binding affinity, or in vitro bioactivity, but had significantly enhanced in vivo potency and half-life [11]. We also studied the production of rhEPO in mammalian cells and observed that hyperglycosylated rhEPO (HGEPO) and dHuEPO have higher erythropoietic activity than wild-type rhEPO, both in vitro and in vivo [12-14]. Transgenic (tg) animals are an attractive alternative to cell cultures for high-level, low-cost production of proteins. The mammary gland is the most reasonable organ for the production of recombinant proteins from transgenic organisms [15,16] and is suitable for synthesis of large amounts of protein that can be easily collected without causing harm to the animal [17]. Attempts have been made to obtain transgenic mice showing enhanced expression of the monomeric form of EPO [18-20]. We have also produced transgenic pigs expressing hEPO protein in the mammary gland, and showed that the purified hEPO had erythropoietic activity [21]. However, there has been no report on the generation of transgenic mice expressing the dHuEPO form. In the present study, we produced tg mice expressing dHuEPO, which was constructed by linking 2 human EPO molecules using a 2-amino acid peptide linker. dHuEPO tg mice developed excessive erythrocytosis that led to short lifespan, debility, and abnormal splenomegaly. Further, by microarray analysis, we have identified 2,672 genes associated with splenomegaly. Methods Construction of the dHuEPO gene The N-terminal EPO domain of the human EPO dimerencoding construct was amplified by polymerase chain reaction (PCR) with a plasmid containing the human EPO cDNA [12] using the primers EPO 1 (5-TGG TCG ACA CCA TGG GGG TGC ACG AAT GTC CT3), which contains the SalI site at the 5 end, and EPO 2 (5-AGG ATA TCT CTG TCC CCT GTC CTG CAG GC-3), which contains the Asp-Ile ligation site that was used to ligate 2 EPO molecules. With the exception of the stop codon, the complete EPO open-reading frame is present in this domain. The C-terminal EPO domain was constructed using the primers EPO 3 (5-ATG ATA TCG CCC CAC CAC GCC TCA TC-3), which contains the Asp-Ile ligation site and in which the signal sequence was removed, and EPO 4 (5-TAC TCG AGT TCA TCT GTC CCC TGT CCT GCA-3), which contains the SalI site at the 3 end. This (...truncated)