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Generation of mice derived from embryonic stem cells using blastocysts of different developmental ages

Laboratory for Genomic Reprogramming, Center for Developmental Biology, RIKEN, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan

Correspondence should be addressed to H Ohta; Email: ohta{at}cdb.riken.jp

	Abstract

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We previously showed that increasing the cell number of host tetraploid (4n) embryos by aggregating multiple 4n embryos at two to four-cell stages can improve the birthrate of mice from embryonic stem cells (ES mice). In the present study, we assessed whether in vitro aged blastocysts (e.g., E4.5 or E5.5), where their cell number also increased with development, can be used as hosts for generating ES mice. As expected, the cell number of in vitro aged 4n blastocysts increased with development, i.e., 26.5±2.4, 49.6±8.4, and 84.9±20.9 cells for E3.5, E4.5, and E5.5 respectively. Three independent ES cell lines were injected into 4n aged blastocysts, and their developmental ability was compared with that of E3.5 4n blastocysts commonly used for this procedure. We found that the birthrate of ES mice derived from E4.5 blastocysts were comparable with those of mice generated from E3.5 blastocysts. On the other hand, the birthrates decreased when E5.5 blastocysts were used. These results suggest that not only the cell number but also developmental age is important for producing ES mice. We also discuss a comparison of the present findings with those of our previous study, where ES mice were generated using an aggregation method employing the same ES cell lines.

	Introduction

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The production of mouse chimeras is a common step in the establishment of genetically modified mouse strains. Chimeric mice are usually produced by injecting or aggregating embryonic stem (ES) cells into diploid (2n) host preimplantation embryos (Nagy et al. 2003) composed of modified donor cells and cells derived from the host embryo. The contribution of donor ES cells to the germ line of chimeric mice allows the generation of mouse strains carrying the haplotype of the ES cells. In general, the production of a mutant strain via this procedure is a time-intensive task, taking more than 12 months before adult mutants can be analyzed. To accelerate the production of mutant mouse lines, a tetraploid (4n) embryo complementation assay has also been established (Nagy et al. 1990, 1993). Since 4n embryos are not capable of completing normal development independently (Kaufman & Webb 1990, Eakin & Behringer 2003), ES cells aggregated with 4n embryos develop into conceptuses in which embryonic lineages are derived entirely from ES cells and extra-embryonic lineages arise largely from the 4n component (Nagy et al. 1990, 1993). Mice derived from ES cells (ES mice) show normal phenotypes, growth, and fertility (Nagy et al. 1993), indicating that ES mice can be used as founders for establishing mouse lines. Because all germ cells in ES mice are derived from ES cells, germ line transmission of ES cells is easier in ES mice than in 2n chimeric mice. Thus, producing ES4n embryos would be effective for analyzing gene function in vivo.

Although the methodology for producing ES mice derived from ES cell lines was described more than a decade ago (Nagy et al. 1990, 1993), its application remains limited due to the extremely low frequency at which viable ES mice are recovered (Nagy et al. 1990, 1993). We previously used a technique where increasing the cell number of host 4n embryos enhanced the production of ES mice (Ohta et al. 2008). The important advantage gained from the improvement of this technique, which was achieved by increasing the cell number of host 4n embryos, was that it could be applied to any ES cell lines such as those commonly used for gene targeting. However, additional host 4n embryos are required in order to reconstruct ES4n chimeric embryos. If this required number can be reduced, the production of ES mice will become easier and more efficient than our previous procedure.

In the present study, we increased the cell number of host 4n embryos by prolonged in vitro culturing, and assessed the functional ability of these in vitro aged blastocysts to generate ES mice.

	Results

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Characterization of in vitro aged blastocysts
We initially characterized 4n blastocysts at different developmental ages (E3.5, E4.5, and E5.5) before the in vitro aged blastocysts were used to produce ES mice. We examined the blastocoel formation of 4n aged blastocysts, and the trophectoderm (TE) and inner cell mass (ICM) cell numbers of 4n aged blastocysts. At E3.5, approximately half of the 4n embryos were formed blastocoel (Fig. 1A: Table 1); therefore, we used and analyzed blastocoel forming E3.5 embryos in the present study. On the other hand, almost all 4n embryos at E4.5 and E5.5 formed a blastocoel (Fig. 1B and C; Table 1).

View larger version (51K): [in this window] [in a new window]   Figure 1 Blastocoel formation of 4n preimplantation embryos of different developmental ages. (A–C) Light micrographs of 4n preimplantation embryos of different ages; (A) E3.5 embryos, (B) E4.5 embryos, (C) E5.5 embryos. Scale bars=200 µm.

View larger version (39K): [in this window] [in a new window]   Figure 2 Immunostaining of 4n blastocysts of different developmental ages with CDX2 or POU5F1 antibodies. The 4n blastocysts of different developmental ages were stained with propidium iodide (PI; A–D) and CDX2 (A'–C'). Merged photographs are shown in (A''–D''); (A–A'') E3.5 blastocysts; (B–B'') E4.5 blastocysts; and (C–C'') E5.5 blastocysts. In D–D'', E5.5 blastocysts were stained with PI (D) or POU5F1 antibody (D'). (A–C'') Stacked serial images of the blastocysts obtained by confocal microscopy are shown. (D–D'') Images were taken under fluorescence microscopy. Scale bars=100 µm.

View larger version (67K): [in this window] [in a new window]   Figure 3 Generation of ES mice from E4.5 4n blastocysts. The 129B6F1G1 ES cell line expressing GFP (A and B) was injected into the blastocoel of E4.5 4n blastocysts. (C and D) ES mouse and placenta at 18.5 dpc delivered from 129B6F1G1E4.5 4n embryos showing a light photograph (C) and its fluorescence image (D). High-intensity green fluorescence was found in the progeny. Scale bars=100 µm.

View larger version (47K): [in this window] [in a new window]   Figure 4 Fate of ES cells injected into E4.5 or E5.5 4n blastocysts. ES cells that express GFP (129B6F1 ES cell line) were injected into E4.5 (A–A''') or E5.5 (B–B''') 4n blastocysts. At 24 h after ES cell injection, the blastocysts were stained with anti-POU5F1 antibody that was visualized with red fluorescence. (A and B) DIC images of E4.5 and E5.5 blastocysts. POU5F1 expression (A' and B') and injected ES cells (A'' and B'') are shown. (A''' and B''') Merged images of A' and A'', B' and B''. Arrows indicate endogenous ICM. In B''', ES cells are indicated as an arrowhead. (C–D''') Primitive endoderm formation by E4.5 and E5.5 4n blastocysts. (C and D) DIC images of E4.5 and E5.5 4n blastocysts. PI (C' and D') and GATA4 expression (C'' and D'') are shown. In C'' and D'', numbers of GATA4 positive 4n blastocysts are shown. (C''' and D''') Merged images of C' and C'', D' and D''.

	Discussion

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In the present study, we examined the ability of in vitro aged blastocysts to generate ES mice. We found that E3.5 and E4.5 4n embryos were similarly efficient in producing ES mice; however, the efficiency was decreased in E5.5 4n embryos (Table 3). Our previous study showed that a larger cell number of host 4n embryos improves the birthrate of ES mice (Ohta et al. 2008). In this report, the cell number of host 4n embryos was increased by aggregating multiple host 4n embryos at the two to four-cell stages, i.e., three two to four-cell stage 4n embryos were aggregated to generate one host 4n embryo. Although the E5.5 4n embryos showed an increase in cell numbers (Table 2) similar to the previous study, the use of E5.5 4n embryos was ineffective in producing ES mice. These results suggest that not only the cell number but also the host embryonic stage is important for generating ES mice.

As stated above, the E5.5 4n blastocysts were inefficient at producing ES mice, even though the cell numbers in the blastocysts were increased (Tables 2 and 3). Interestingly, the implantation and developmental abilities of the E5.5 4n blastocysts were similar to those of the E3.5 4n or E4.5 4n blastocysts (Table 5), which indicate that the low efficiency of E5.5 4n blastocysts is not attributable to dysfunctions in implantation or subsequent development. To further this investigation, we carried out a cell tracing experiment to examine whether the ES cells injected into E5.5 4n blastocysts could contribute to the ICM. We found that the ES cells injected into E5.5 4n blastocysts made a relatively weak contribution to the ICM (Table 6 and Fig. 4A–B'''). These results suggest that the primary cause of inefficient production of ES mice from E5.5 blastocysts is inefficient homing of injected ES cells to the ICM, possibly due to the ICM status of the E5.5 4n blastocysts. As the ICM of E5.5 4n blastocysts has already differentiated into two lineages (epiblast and primitive endoderm; Fig. 4C–D'''), the formation of primitive endoderm may inhibit the integration of injected ES cells into the epiblast. Thus, although the cell numbers of the host 4n embryos are important to produce ES mice (Ohta et al. 2008), there is a time window for the integration of the ES cells into the host 4n epiblast. A potential strategy to increase the ES mouse producing ability of host 4n blastocysts is the addition of insulin to the culture medium, as it has been known to increase the cell numbers of 4n blastocysts (Koizumi & Fukuta 1996).

Our results showed that E4.5 4n blastocysts were similarly efficient as those of E3.5 in generating ES mice (Table 3). The advantage in using E4.5 blastocysts is that they can be injected at a more convenient time than E3.5 blastocysts. As shown in Fig. 1 and Table 1, almost all E4.5 4n blastocysts formed a blastocoel, whereas morula-stage embryos were still present in E3.5 4n embryos. Although some of the ES mice derived from E4.5 4n embryos showed abnormalities such as abdominal hernia or large offspring syndrome (Table 4), the frequency of these defects was similar to that in ES mice generated from E3.5 4n embryos (Table 4), indicating that ES mice can be produced from both E4.5 and E3.5 4n embryos. Thus, our results demonstrate that the use of E4.5 embryos is effective in generating ES mice.

In general, ES mice can be produced using two methods (Nagy et al. 2003): injection of ES cells into 4n blastocysts (Wang et al. 1997) or aggregation of ES cells with 4n four-cell stage embryos (Nagy et al. 1990, 1993). In our previous study, we demonstrated that the use of multiple 4n host aggregates (e.g., three 4n embryos) is more effective in generating ES mice than the use of one or two 4n host embryos (Ohta et al. 2008). In this report, the birthrate of ES mice using E14 and 129B6F1G1 strains with three 4n embryos was 14.3 and 9.3% respectively (Ohta et al. 2008). As this birthrate was calculated based on the number of transferred ES3x4n embryos, that estimated from the number of embryos used was 4.8% for E14 and 3.1% for 129B6F1G1. In the present study, the birthrate of ES mice via blastocyst injection using E3.5 and E4.5 embryos was 3.8% for E14 and 1.8% for 129B6F1G1 (Table 3). Thus, the production of ES mice based on the number of embryos employed using blastocyst injection is similar to that using multiple 4n embryos. These two techniques can be selectively used depending on the research purpose; for example, blastocyst injection is effective for assessing donor cell function since micromanipulation allows the selection of donor cells. On the other hand, the use of 3x4n embryos is effective in producing ES mice because the procedure is easier to perform. Recently, another procedure for producing ES mice have been reported, in which ES cells were injected into 2n host four- to eight-cell stage embryos (Poueymirou et al. 2007). Although we could not compare the efficiencies of this and the above-described procedures, this method may also be suitable for the production of ES mice.

	Materials and Methods

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Mice
ICR mice were purchased from SLC (Hamamatsu, Japan). All animal experiments conformed to the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Committee of the Laboratory Animal Experimentation of the RIKEN Kobe Institute.

ES cell lines
The ES cell line E14 (Hooper et al. 1987) was derived from the inbred mouse strain 129/Ola in 1985 by Dr Martin Hooper in Edinburgh, Scotland, and obtained through Dr Peter Mombaerts (Rockefeller University). 129B6F1G1 and GR14 are nuclear transfer-derived ES (ntES) cell lines (Wakayama et al. 2001) previously established in our laboratory using Sertoli cells from a 129B6F1 background with GFP (Ohta & Wakayama 2005) and tail tip cells of a male mouse (129BDF2; Wakayama et al. 2005) respectively, as donors for nuclear transfer. Karyotype analysis revealed that the ES cell lines used had the normal karyotype at the following percentages (number of metaphases with normal karyotype in parentheses): 46% (12/26) for E14; 35% (9/26) for 129B6F1G1; and 54% (14/26) for GR14.

ES cell culture conditions
ES cells were grown in KNOCKOUT Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 20% heat-inactivated fetal bovine serum (FBS; Sigma–Aldrich), 1000 units of leukemia inhibitory factor/ml (Invitrogen), and the following reagents: 1% penicillin–streptomycin (Invitrogen); 1% L-glutamine (Specialty Media, Phillipsburg, NJ, USA); 1% non-essential amino acids (Specialty Media); 1% nucleosides (Specialty Media); and 1% β-mercaptoethanol (Specialty Media). Cells were cultured in a gelatin-coated dish in feeder-free conditions and split 1:5 or 1:10 every 48 h.

Generation of ES4n embryos
Blastocysts with an ICR background were used as hosts for 4n embryos. Briefly, superovulated ICR females were mated with ICR males, and two-cell stage embryos were collected from the oviducts at 1.5 dpc. To generate 4n embryos, two-cell stage embryos were electrofused using an electro cell fusion system (Model LF101; Nepagene, Chiba, Japan). Fused embryos were selected and cultured in CZB medium (Chatot et al. 1989). Each ES cell line was used within three passages in our experiments. Approximately, 15 ES cells were injected into the blastocysts using a piezo-actuating micromanipulator, using essentially the same procedure as that reported by Wang et al. (1997). All blastocysts injected with ES cells were transferred to pseudopregnant ICR females at 2.5 dpc (10–16 chimeric embryos/mouse), which were analyzed at 18.5 dpc. Body and placenta weight were assessed in newborn ES mice. ES mice with open eyelids, abdominal hernia, or large offspring (body weight more than 2.0 g) were categorized as abnormal pups.

Assessment of cell numbers of blastocysts
The total and TE cell numbers of blastocysts were determined. Propidium iodide (PI) staining and immunostaining for CDX2 were used to indicate the total and TE cell numbers respectively. The cell number for ICM was roughly estimated as the TE cell number subtracted from total cell number (total TE). Blastocysts were fixed with 4% paraformaldehyde, washed with PBS containing 1% BSA, and incubated with anti-CDX2 MAB (1:200; BioGenex, San Ramon, CA, USA). The primary antibody was visualized with goat anti-mouse IgG conjugated with Alexa Fluor 488 (Molecular Probes, Eugene, OR, USA). After immunostaining for CDX2, blastocysts were incubated in PBS containing 1 µg/ml PI. Serial confocal images were taken using fluorescence confocal microscopy (Yamagata et al. 2005), and three-dimensional images of blastocysts were reconstructed using MetaMorph software (Universal Imaging Co., Downingtown, PA, USA). More than ten blastocysts were stained and the total (PI-positive) and TE (CDX2-positive) cell numbers were determined. Immunostaining for POU5F1 or GATA4 was performed employing the same procedure for the CDX2 staining using an anti-POU5F1 MAB (C-10; 1:200; Santa Cruz Biotechnology Inc., CA, USA; catalog no. sc-5279) or anti-GATA4 polyclonal antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA; catalog no. sc-9053).

Statistical analysis
The Fisher's exact probability test (Tables 3, 5, and 6) and Student's t-test (Table 2) were performed. P<0.05 was considered statistically significant.

	Declaration of interest

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The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

	Funding

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This work was supported by grants for Scientific Research in Priority Areas (15080221) and the Project for the Realization of Regenerative Medicine (research field: technical development of stem cell manipulation) to T W by the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

	Acknowledgements

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We thank Dr Kazuo Yamagata for assistance with confocal microscopy. We also thank the Laboratory for Animal Resources and Genetic Engineering for housing the mice used in the study.

Received 30 April 2008
First decision 28 May 2008
Revised manuscript received 15 July 2008
Accepted 28 August 2008

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This Article Abstract Full Text (PDF) All Versions of this Article: 136/5/581    most recent REP-08-0184v1 Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ohta, H. Articles by Wakayama, T. Search for Related Content PubMed PubMed Citation Articles by Ohta, H. Articles by Wakayama, T.