Generation of transgenic mouse line expressing Kusabira Orange throughout body, including erythrocytes, by random segregation of provirus method
Introduction
Red/Orange fluorescent proteins such as Discosoma red (DsRed) (excitation/emission wave lengths 558/583 nm), tdTomato (554/571 nm), mCherry (587/610), and Kusabira Orange (KuO) (548/561 nm) [1], [2], [3] are widely used to visualize cells, to track cells and their progeny, or to quantify molecules in living cells. Whilst high level expression of oligomeric red/orange fluorescent proteins may induce growth defects and instability of fluorescent-protein expression, monomeric red fluorescent protein (mRFP; 584/607) induces almost no toxicity [4], [5]. Although previously immunogenicity of the enhanced green fluorescent protein (EGFP) [6], [7] was reported, we found that mouse hematopoietic stem cells (mHSCs) transduced with humanized Kusabira Orange (KuO) behaved like wild-type mHSCs in recipient mice after transplantation [8]. Furthermore, no toxicity was apparent in KuO-expressing cloned pigs generated by nuclear transfer of KuO-transduced fibroblasts [9]. These results indicate that KuO is an appropriate fluorescent protein for biological research, including research into hematopoiesis.
Moloney murine leukemia virus (MoMLV)-based retroviral vectors have been most commonly used for gene transduction. MoMLV vectors, however, show low level transcriptional activity in embryonic or hematopoietic stem cells, due to promoter methylation or to promoter binding of transcriptional inhibitory factors [10], [11], [12].
We have improved the retroviral vector: GCDsap by long terminal repeat (LTR) modification to reduce susceptibility to promoter methylation. Using GCDsap carrying enhanced green fluorescent protein (EGFP), we successfully generated mouse embryonic stem cells (mESCs) expressing high levels of EGFP and, from those cells, EGFP transgenic mice (retro-GFP mice). EGFP was stably expressed even in F1 progeny (after germline transmission) [13]. In these retro-GFP mice, EGFP was expressed throughout the body, including leukocytes and platelets, but was not expressed in erythrocytes. This may owe to the location of the retroviral integration site, on the closed chromatin region in erythroid precursors. This lack of expression of EGFP in erythroid-lineage elements is a major restriction upon use of retro-GFP mice. As far as we know, no fluorescent-protein transgenic mouse is described in which fluorescent protein is expressed in all hematopoietic lineages, including erythroid precursors and erythrocytes.
We generated KuO-expressing transgenic mice derived from mESCs transduced by GCDsap carrying KuO (GCDsapKuO). In these mice, KuO was highly expressed in both solid tissues and hematopoietic cells, including HSCs and the five blood lineages (ending in neutrophils/macrophages, T-cells, B-cells, platelets, and erythrocytes). Finding the open chromatin region in Ter119 expressing-erythroid precursors was achieved through random segregation of provirus (RSP) after germline transmission from GCDsapKuO-infected mESCs that carry multiple proviruses. KuO transgenic mice showed no obvious abnormality in development, exhibited no tendency to hemorrhage, and did not develop tumors.
Section snippets
Mice
C57BL/6 mice congenic for the Ly5 locus (B6-Ly5.2) mice were purchased from Japan SLC (Shizuoka, Japan) and Nihon Clea (Tokyo, Japan). B6-Ly5.1 mice and recombination activating 2 knockout (Rag2 KO) mice were bred and maintained at Sankyo Labo Service (Tokyo, Japan). All procedures were approved by the Animal Care and Use Committee, University of Tsukuba, and by the Institute of Medical Science, University of Tokyo.
Retroviral vectors
The retroviral vector [GCD/huKO(KuO)] was constructed by insertion of KuO cDNA
KuO-expressing chimeric mice and progeny from transduced ES cells
To use the RSP method (Fig. 1A) of finding open chromatin region in all hematopoietic cells, including erythroid precursors, we initially generated mESCs carrying multiple provirus copies [14]. We infected mESCs with GCDsapKuO (Fig. 1B) by following three protocols: Single infection at multiplicity of infection (MOI) of 10, duple infection at MOI of 40, and duple infection at MOI of 40 with low-speed centrifugation. Efficiencies of infection were 73%, 89%, and 94% respectively (Fig. 1C). We
Discussion
In this study we used the RSP method to generate KuO transgenic mice in which KuO was expressed in all hematopoietic cells, including erythrocytes. RSP technique requires generation of ESCs carrying randomly integrated provirus in high copy number. To achieve this, we performed retroviral infection at high MOI with low speed centrifugation, thereby generating ESCs carrying more than 15 integrated copies of provirus. We then backcrossed KuO transgenic mice and chromosome segregation was utilized
Acknowledgments
We thank Dr. A.S. Knisely (Institute of Liver Studies, King’s College Hospital) for critical reading of the paper. This work was supported by grants from JST, the Ministry of Education, Culture, Sport, Science, and Technology, Japan, and in part by the Monbu-Kagaku-Sho (MO) and the Cooperative Study Program of National Institute for Physiological Sciences, Japan.
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In vivo clonal analysis of aging hematopoietic stem cells
2020, Mechanisms of Ageing and DevelopmentCitation Excerpt :However, as CD45 is not expressed on erythrocytes and platelets, use of this system precludes studies of HSC differentiation potential along these two lineages. Generation of a transgenic mouse line expressing Kusabira Orange throughout the body, including on erythrocytes and platelets, has overcome this issue, and again in combination with single cell transplantation has afforded identification of five-lineage potential in HSCs, including the erythrocyte and platelet lineages (Hamanaka et al., 2013). These approaches to quantitate reconstitution of erythrocytes and platelets have identified repopulating or self-renewing cells with limited lineage potential.
Generation of Vascular Endothelial Cells and Hematopoietic Cells by Blastocyst Complementation
2018, Stem Cell ReportsCitation Excerpt :The iPSCs were generated by introducing three reprograming factors (Oct-3/4, Sox-2, and Klf-4) and EGFP in all-in-one inducible lentiviral vector (Ai-LV) (Yamaguchi et al., 2012). KuO miPS were generated from MEFs of KuO mice by introducing three reprograming factors (Oct-3/4, Sox-2, and Klf-4) in Ai-LV and CAG huKO IRES Puro (Hamanaka et al., 2013). riPST1-3 were generated from rat embryonic fibroblasts of Wistar rat by introducing three reprograming factors (Oct-3/4, Sox-2, and Klf-4) in Ai-LV (Hamanaka et al., 2011).
Large-Scale Clonal Analysis Resolves Aging of the Mouse Hematopoietic Stem Cell Compartment
2018, Cell Stem CellCitation Excerpt :To directly interrogate the functional characteristics of individual young and aged CD34−KSL cells, we performed single-cell transplantation into lethally irradiated young mice. We initially transplanted single CD34−KSL cells from young (8- to 12-week-old) and aged (20- to 24-month-old) Ly5.1 Kusabira Orange (KuO) mice (Hamanaka et al., 2013) into a total of 76 lethally irradiated young Ly5.2 mice, together with 2 × 105 competitor cells from Ly5.1/Ly5.2-F1 mice (Figure 1C). As we observed significant changes in CD150 and CD41 expression within the CD34−KSL population with age, we also transplanted single CD150+CD41−CD34−KSL (fraction 1), CD150+CD41+CD34−KSL (fraction 2), and CD150−CD41−CD34−KSL (fraction 3) cells from young and aged mice into a total of 370 lethally irradiated Ly5.2 young mice.
- 1
Present address: Institute of Molecular Medicine and Max-Planck-Research Group on Stem Cell Aging, University of Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany.
- 2
Present address: Department of Cell Differentiation, The Sakaguchi Laboratory of Developmental Biology, School of Medicine, Keio University, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan.