Generation of transgenic mouse line expressing Kusabira Orange throughout body, including erythrocytes, by random segregation of provirus method

https://doi.org/10.1016/j.bbrc.2013.05.017Get rights and content

Highlights

  • We have generated KuO transgenic mouse line by random segregation of provirus method.

  • KuO transgenic mouse line cells carry only one proviral integration site.

  • KuO transgenic mouse line cells stably express KuO in erythrocytes and platelets.

Abstract

Fluorescent-protein transgenic mice are useful for obtaining marked somatic cells to study kinetics of development or differentiation. Fluorescence-marked hematopoietic stem cells in particular are commonly used for studying hematopoiesis. However, as far as we know, no transgenic mouse line is described in which a fluorescent protein is stably and constitutively expressed in all hematopoietic cells, including erythrocytes and platelets. Using the random segregation of provirus (RSP) method, we generated from retrovirally transduced mouse embryonic stem cells a transgenic mouse line expressing a red/orange fluorescent protein, Kusabira Orange (KuO). KuO transgenic mouse line cells carry only one proviral integration site and stably express KuO in all hematopoietic-lineage elements, including erythrocytes and platelets. Moreover, bone-marrow transplantation in KuO transgenic mice demonstrated normal hematopoieisis. KuO transgenic mice likely will prove useful for study of hematopoiesis that includes erythropoiesis and megakaryopoiesis.

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.

References (18)

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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.

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