First published online May 8, 2008
Stem Cells
Vol. 26 No.
7
July 2008, pp.
1883
-1890
doi:10.1634/stemcells.2008-0164; www.StemCells.com
© 2008 AlphaMed Press
Efficient Production of Mice from Embryonic Stem Cells Injected into Four- or Eight-Cell Embryos by Piezo Micromanipulation
Junjiu Huanga,b,
Kai Denga,
Haojia Wua,
Zhong Liua,
Zhisheng Chena,
Shanbo Caoa,
Lingjun Zhoua,
Xiaoying Yec,
David L. Keefeb,
Lin Liub,c
aCollege of Life Sciences, Sun Yat-Sen University, Guangzhou, China;
bDepartment of Obstetrics and Gynecology, University of South Florida College of Medicine, Tampa, Florida, USA;
cCollege of Life Science, Nankai University, Tianjin, China
Key Words. Embryonic stem cells • Piezo microinjection • Mice • Pluripotency
Correspondence:
Correspondence: David L. Keefe, M.D., Department of Obstetrics and Gynecology, University of South Florida College of Medicine, 12901 Bruce B. Downs Boulevard, Tampa, Florida 33612, USA; e-mail: dkeefe{at}health.usf.edu; or Lin Liu, Ph.D., University of South Florida College of Medicine, 12901 Bruce B. Downs Boulevard, Tampa, Florida 33612, USA; e-mail: liutelom{at}yahoo.com
Received on February 19, 2008;
accepted for publication on April 28, 2008.
First published online in STEM CELLS EXPRESS May 8, 2008.
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ABSTRACT
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The conventional method for producing embryonic stem (ES) cell-derived knockout or transgenic mice involves injection of ES cells into normal, diploid blastocysts followed by several rounds of breeding of resultant chimeras and thus is a time-consuming and inefficient procedure. F0 ES cell pups can also be derived directly from tetraploid embryo complementation, which requires fusion of two-cell embryos. Recently, F0 ES cell pups have been produced by injection of ES cells into eight-cell embryos using a laser-assisted micromanipulation system. We report a simple method for producing F0 ES cell germline-competent mice by piezo injection of ES cells into four- or eight-cell embryos. The efficiency of producing live, transgenic mice by this method is higher than that with the tetraploid blastocyst complementation method. This efficient and economical technique for directly producing F0 ES cell offspring can be applicable in many laboratories for creating genetically manipulated mice using ES cell technology and also for stringent testing of the developmental potency of new ES cell or other types of pluripotent stem cell lines.
Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION
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The development of gene targeting technology using ES cells has been very useful for understanding gene function in vivo [1–7] and has contributed significantly to biomedical research [8, 9]. The conventional method for producing ES cell-based transgenic mice involves injection of ES cells into normal diploid embryos, mostly blastocysts [10, 11], followed by several rounds of breeding of resultant chimeras. An alternative method uses tetraploid embryo complementation, which only contributes to placental development. This method generates ES cell gene-targeted mice directly, thus avoiding the requirement to breed chimeras [12–14]. ES cell pups have been produced from early-passage ES cells derived from hybrid mice, but the efficiency of this method is low [12]. Similar to previous observations that the eight-cell embryo injection method produced high viable germline chimeras [15], a recently reported technique involving laser-assisted injection of ES cells into eight-cell stage embryos produced F0 ES pups with full germline transmission [16] and was easier and more efficient than thetetraploid complementation method. The tetraploid complementation method requires fusion of two-cell stage embryos by electroporation or chemical methods, and tetraploid embryos are less viable than normal diploid embryos. Pups produced by tetraploid complementation also suffer from nonspecific lethality and congenital abnormalities, complicating phenotypic analyses [16]. In the laser-assisted method, the laser is used to first cut the zona pellucida (ZP) and then the ES cells are injected into eight-cell stage embryos through a bevelled injection pipette. This method requires laser equipment and extensive experience and can take a longer time during micromanipulation, which may impact negatively on subsequent embryonic development, particularly for gene-manipulated ES cells.
To simplify the injection procedure and reduce the cost, we used the piezo micromanipulation method, which originally was successfully used for intracytoplasmic sperm injection and somatic cell cloning [17, 18] and injected ES cells into four- or eight-cell embryos cultured in vitro. We successfully produced pups directly from ES cells, and these pups show full germline transmission.
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METHODS
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Embryos and Recipient Mice
All mice were housed in isolator units, maintained on a 10-hour dark/14-hour light cycle and were cared for on the basis of approved animal protocols by the Institutional Animal Care Committee. For the production of four- or eight-cell embryos, 4- to 8-week-old outbreed Kunming (KM) albino females were superovulated by intraperitoneal injection of pregnant mare serum gonadotrophin (5 IU, Calbiochem, San Diego, http://www.emdbiosciences.com) followed 46–48 h later by injection of human chorionic gonadotrophin (5 IU) and mated with males with proven fertility. Females were screened for vaginal plugs the following morning (0.5 day post coitum, [dpc]), and fertilized embryos were collected and cultured in groups of 20–30 in a 50-µl droplet of potassium simplex optimization medium (KSOM) with amino acids (KSOMAA) [19], overlaid with embryo-tested mineral oil in humidified atmospheres of 6.5% CO2 at 37°C. ICR females were mated with vasectomized KM males and used as recipients for embryo transfer at 2.5 dpc. Unless otherwise specified, all reagents were obtained from Sigma-Aldrich (St. Louis, http://www.sigmaaldrich.com).
ES Cell and Culture
F1 (the first) and F5 (the fifth) ES cell lines were derived in our laboratory from fertilized embryos of 129XB6F1 mice (with typical phenotype of agouti in coat color and black eyes) by methods detailed previously [20]. Briefly, day 3.5 blastocysts were placed onto mitomycin C-treated mouse embryonic fibroblast feeder cells. Embryos outgrew and developed to clones several days after culture in ES cell medium, composed of 80% knockout Dulbecco's modified Eagle's medium (KDMEM) (10829018; Gibco, Gaithersburg, MD, http://www.gibcobrl.com) supplemented with 20% fetal bovine serum, 50 units/ml penicillin, 50 µg/ml streptomycin, 1 mM glutamine, 1% nonessential amino acid stock, 0.1 mM β-mercaptoethanol, and 1,000 units/ml ESGRO leukemia inhibitory factor (Chemicon International Inc., Temecula, CA, http://www.chemicon.com). Typical clones were picked, digested, and passaged. The ES cell lines were initially checked for pluripotency by expression of molecular markers specific for ES cells, teratoma formation, and successful production of chimera and germline transmission. Karyotypes of the two cell lines were XY.
Transgenic ES Cell Lines
Nanog gene promoter fragment was amplified from mouse genome by polymerase chain reaction (PCR), using primers 5'-TAATACCGGTGTGATGGCGAGGGAAGGGATTT-3' and 5'-ATCTGGTACCACCTCTTCGCTCGGATCTTTCACC-3'. The resulting 5,045-base pair (bp) product was then inserted into the promoterless vector pEGFP-1 (Clontech, Palo Alto, CA, http://www.clontech.com) between KpnI and AgeI sites (Fig. 4A). Both Nanog promoter-enhanced green fluorescent protein (EGFP) and pCX-EGFP (chicken β-actin promoter-EGFP [21]) plasmids were linearized and introduced into an ES cell line (XY) derived from 129XB6F1 mice by transfection (Lipofectamine 2000; Invitrogen, Carlsbad, CA, http://www.invitrogen.com). Neomycin (G418; Sigma-Aldrich) selection was performed 2 days after transfection to obtain stable transgenic ES cell lines.
Generation of F0 ES Cell Pup
The mouse ES cells were introduced into early embryos by piezo micromanipulation as described [17, 18, 22]. Fertilized embryos were cultured in KSOMAA in vitro until 2.0 and 2.5 dpc. For easy recognition of the phenotypes of the resultant fetuses and pups, eight- or four-cell embryos from albino KM mice with white eyes were used as recipients for supporting development of donor ES cells. The setup for injection of ES cells into eight- or four-cell embryos before obvious compaction using a Leica micromanipulator and piezo micromanipulator (PMM-150FU; Prime Tech Ltd., Tsuchiura-shi, Ibaraki-ken, Japan, www.primetech-jp.com) was similar to that for blastocyst injection. Mercury in a 3-mm length was back-loaded into an injection pipette. The blunt end of the pipette had a bore of approximately 15 µm in diameter. The mercury was pushed through the shoulder to near the tip to empty the air in the pipette, which was then washed three times in 10% polyvinylpyrrolidone (PVP)-PBS solution and in Hepes-buffered KSOM (HKSOM) medium, respectively. Roughly 1 mm HKSOM was sucked into the pipette to keep ES cells away from mercury, and dozens of ES cells were then sucked for injection and maintained approximately 200 µm away from the pipette tip. Damage to cell membranes may occur if the cells are very close to the tip when the piezo pulses are applied. The pipette was positioned near the ZP above the perivitelline space between two embryonic cleavage cells. The ZP was pierced by the pipette immediately after application of two to three electropulses in a very short time generated from the piezo manipulator, and then 8–10 ES cells were injected between the embryonic cells under the ZP, followed by slow withdrawal of the pipette. All embryos injected with ES cells were cultured in KSOMAA at 37°C in an atmosphere of 6.5% CO2 until 3.5 dpc. Healthy embryos (morula and blastocysts) that had progressed beyond the eight-cell stage were transferred into the uterine horns of pseudo-pregnant ICR females by the standard procedure. Tetraploid embryos were produced from fusion of two-cell embryos by electroporation. Twenty to 25 ES cells were injected into KM tetraploid blastocysts by piezo micromanipulation, as described [22]. Compared with injection of 30–40 tetraploid blastocysts per hour by using the piezo method, 60–70 four- or eight-cell embryos can be injected within an hour. Injected blastocysts then were transferred into the uterine horns of pseudo-pregnant ICR females.
Fluorescence Microscopy
EGFP cells, embryos, and fetuses were visualized at excitation wavelengths of 488 nm with a Leica inverted fluorescence microscope.
Integrity Test of ES Cells and Embryos
The four- and eight-cell stage embryos were injected with 10 ES cells using the piezo micromanipulator. ES cells or ES cells with injected embryos by piezo pulses were cultured in 50-µl KSOM or ES cell medium drops in Petri dishes for various time, incubated with 15 µg/ml propidium iodide (PI) for 20 min, washed twice in KSOM or ES cell medium, and then imaged using a rhodamine filter under a Zeiss fluorescence microscope. Viable cells show negative PI staining, whereas damaged cells exhibit positive PI staining. Embryos cultured in vitro for 48 and 72 h without treatment were used as negative controls, and embryos treated with 0.1% Triton X-100 and 0.1% PVP-PBS buffer for 3 min were used as positive controls.
PCR and Microsatellite Analysis
Genomic DNA was extracted from tissue samples using the E.Z.N.A Tissue DNA Kit (Omega Bio-tek., Inc., Norcross, GA, www.omegabiotek.com). DNA from ES cell pups was analyzed by standard microsatellite and PCR analysis with the following primers: D12Mit233 (148 bp), sense primer 5'-AGGCTCACAGAAAAAGTGAACC-3' and antisense primer 5'-TGAAGCAGTCTGACCAGGTG-3'; Nanog-EGFP plasmid (1,890 bp), sense primer 5'-CGGCCCTTCCCTCTCTGCTTATACA-3' and antisense primer 5'-CGCGCTTCTCGTTGGGGTCTT-3' in a total volume of 20 µl. Amplification was performed in a gradient PCR device under the following conditions: 5 min at 95°C and 35 cycles of 30 s at 95°C, 30 s at 61°C, and 30 s at 72°C, followed by 10 min at 72°C.
Statistical Analysis
Data as percentages were analyzed by 
2 test. Significant differences within the column were defined as p < .05 or .01.
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RESULTS
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ES Cell Mice Produced from ES Cells Injected into Eight- or Four-Cell Stage Recipient Embryos by Piezo Micromanipulation
We attempted to obtain ES cell mice from two normal ES cell lines (F1 ES cells and F5 ES cells) by injection into eight- or four-cell stage embryos using a piezo manipulator (Fig. 1A, 1B). The number of implantations and newborns produced by eight-cell embryo injection were lower than (p < .05) those produced by four-cell embryo injection. Pups produced by injection of ES cells into either eight- or four-cell embryos can be categorized into three types: host-derived, chimera, and ES cell mice (Table 1; Fig. 1C, 1D), but the percentage of newborns with black eyes did not differ between the two types of embryo injections. Based on visualization of black eyes in the newborn, an average of 70% of pups was directly produced from ES cells. Microsatellite analysis of the DNA bands from tails of black-eye pups using D12Mit233 primers showed that more pure ES cell pups were produced using eight-cell embryos as recipients (62.5%: five ES pups of eight black-eye pups analyzed) (Table 1; Fig. 1G) compared with use of four-cell embryos (23.1%: 3 ES pups of 13 black-eye pups analyzed) (Table 1; Fig. 1H). Four ES cell pups from injection into eight-cell embryos developed into adults and exhibited full germline transmission (Fig. 1E). One ES cell mouse, from injection into a four-cell embryo, developed into an adult and had full germline transmission (Fig. 1F). Therefore, eight-cell embryos appeared to provide a better host for supporting ES cell development. Microsatellite analysis using D12Mit233 primers showed that most cells in the placenta were derived from ES cells when four-cell embryos served as recipients, but fewer cells were derived from ES cells when eight-cell embryos served as recipients (Fig. 1I, 1J). Microsatellite analysis of various tissues of ES cell pups demonstrated that ES cell pups were derived from ES cells using either eight- or four-cell embryos as recipients (Fig. 1K–1N).
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Figure 1. Pups produced by injection of embryonic stem (ES) cells into eight- or four-cell recipient embryos. (A, B): Injection of ES cells into eight-cell (A) or four-cell (B) embryos by piezo micromanipulation. Scale bar = 20 µm. (C, D): Newborn mice delivered by cesarean section at embryonic day 19.5 from ES cells after injection into eight-cell (C) or four-cell (D) embryos. Pups with white eyes were from host KM embryos, pups with gray eyes were chimera, and pups with black eyes were from agouti coat color mice. (E, F): ES cell mice and a litter of agouti pups showing germline transmission from ES cells injected into eight-cell (E) or four-cell (F) embryos. Female albino KM mice were used for mating. (G, H): Microsatellite analysis of tail DNA from black-eye pups with eight-cell embryos (G) as recipient (1–4 and 7–8 from F1 ES cells; 5 and 6 from F5 ES cells; 9 gray-eye chimera from F1 ES cells) or four-cell embryos (H) as recipient (3, 4, 12, and 13 from F1 ES cells; 1, 2, and 5–11 from F5 ES cells). (I, J): Microsatellite analysis of placenta DNA from ES injected into eight-cell embryos (I) (1–5 and 8–11 from F1 ES cells; 6 and 7 from F5 ES cells) and four-cell embryos (J) (6–9 from F1 ES cells; 1–5 from F5 ES cells). In (G) 1, 2, and 6–8 bands showed no DNA from KM recipient embryos. In (H) 7, 10, and 12 bands showed no DNA from KM embryos. (K–N): Microsatellite analysis of tissue DNA of pups produced from ES cells injected into either eight-cell embryos ([K]: 1-day-old pup from F1 ES cells; [L]: 3-week-old pup from F5 ES cells) or four-cell embryos ([M]: 2-week-old pup from F1 ES cells; [N]: 1-day-old pup from F5 ES cells). Abbreviations: b, black eyes; ESC, embryonic stem cell; g, gray eyes; KM, Kunming; w, white eyes.
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ES Cells Devoted to Inner Cell Mass and Fetuas
To trace the fate of the ES cells, we produced ES cell fetuses by piezo injection of β-actin promoter-EGFP ES cells into eight- or four-cell embryos. After injection, ES cells preferentially allocated to the inner cell mass, but not to trophectoderm layers when injected embryos developed to blastocysts (Fig. 2A–2C, 2D–2F). Most embryos, however, showed increased cells expressing green fluorescent protein (GFP) fluorescence, whereas few showed reduced expression after injection of ES cells (Fig. 2B, 2E). When cesarean sections were performed on embryonic day (E) 12.5 fetuses after embryo transfer, most fetuses (five of seven) expressed strong GFP fluorescence, regardless of whether eight- or four-cell embryos served as hosts (Fig. 2G, 2I), and only a few (two of seven) exhibited no fluorescence (Fig. 2H, 2J). A small population of EGFP fluorescent cells were observed in the placenta after injection of ES cells into four- or eight-cell embryos.
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Figure 2. Embryos and fetus produced by injection of β-actin promoter-EGFP ES cells into eight- or four-cell recipient embryos. (A–E): Fluorescent embryos from injection of β-actin promoter-EGFP ES cells into either eight-cell ([A]: 0 h; [B]: 24 h; [C]: 56 h) or four-cell ([D]: 0 h; [E]: 24 h; [F]: 58 h) embryos. Scale bar = 20 µm. (G–J): Fetuses produced from injection of β-actin promoter-EGFP ES cells into eight-cell ([G]: green; [H]: no fluorescence) or four-cell ([I]: green; [J]: no fluorescence) recipient embryos by cesarean at embryonic day 12.5. Abbreviations: DIC, differential interference contrast microscopy; E, embryonic; FITC, fluorescein isothiocyanate.
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To test whether the integrity of ES cells injected into recipient embryos were disrupted by piezo pulses, we stained ES cells and embryos with PI and imaged them using a fluorescence microscope. Nonviable cells show PI-positive staining. Less than 5% of ES cells were positive for PI staining in the four- or eight-cell embryos right (0.5 h) after injection, but more ES cells exhibited PI-positive staining after culture in KSOM for 24 h (Fig. 3A; Table 2). KSOM was optimized for development of early embryos and embryos cultured in KSOM for 48 and 72 h as controls showed no PI staining (Fig. 3B). More ES cells exhibited PI-positive staining in the injected four-cell embryos cultured in KSOM for 24 h compared with the injected eight-cell embryos (42% vs. 28%) (Table 2). Notably, no significant differences were observed in the PI staining when injected embryos were cultured in ES cell medium for 24 h (7% vs. 3%, p > .05), suggesting that culture medium could affect the integrity of injected ES cells. Furthermore, similar proportions of ES cells exhibited PI-positive staining during in vitro culture regardless of micromanipulation, indicating that piezo pulses themselves did not compromise viability of ES cells during injection (Table 2; Fig. 3C). These data suggest that ES cells survived from piezo injection manipulation, but some showed decreased viability during culture in KSOM. A longer time in KSOM might lead to reduced integrity of ES cells. This may explain why more chimeras were produced by the four-cell injection method when ES cells remained in the embryos for a longer time compared with the eight-cell injection method.
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Figure 3. Micrographs showing propidium iodide (PI) staining of ES cells after injection into four- or eight-cell embryos or during culture in potassium simplex optimization medium (KSOM) or ES cell medium. (A): Some ES cells show positive PI staining (red) in the four- or eight-cell embryos after piezo injection. (B): Embryos cultured in vitro for 48 and 72 h served as negative or positive (treated with Triton X-100) controls. (C): PI staining of ES cells cultured in KSOM or ES cell medium after piezo pulses. Red, PI positive cells. . Scale bar = 20 µm. Abbreviations: ES-M, embryonic stem cell medium; KS, potassium simplex optimization medium with amino acids.
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Nanog Promoter-EGFP Mice Produced Directly from ES Cells Injected into Eight- or Four-Cell Embryos
We further tested the efficacy of producing transgenic mice using ES cell and gene manipulation technology between eight- or four-cell embryo injection and tetraploid blastocyst injection methods. The Nanog promoter-EGFP plasmid (Fig. 4A) was introduced into ES cell lines derived from 129XB6F1 mice. We attempted to obtain transgenic mice by injection of Nanog promoter-EGFP ES cells into tetraploid blastocysts but failed to produce live adult mice (Table 3). Nearly all newborns died soon after cesarean sections and appeared abnormal (Fig. 4B). However, live pups were produced and survived to adults after Nanog promoter-EGFP ES cells were injected into eight- or four-cell embryos (Table 3; Fig. 4C). The number of implantations produced by tetraploid embryo complementation method were lower than (p < .05) those produced by the four- or eight-cell embryo injection method. However, the percentage of newborns based on implantation was greater by the tetraploid embryo complementation method than by the four- or eight-cell embryo injection method. Although 60%–100% of newborns were derived from ES cells based on the eye color by tetraploid complementation or four- or eight-cell embryo injection, pups from four- or eight-cell embryo injection survived to adulthood and exhibited full germline transmission (Fig. 4C). To confirm reliable specific expression of Nanog in known cells, we also collected and cultured zygotes from KM albino females mated with Nanog promoter-EGFP male mice. Not surprisingly, Nanog promoter-EGFP fluorescence was detected when embryos developed beyond eight-cell stages (Fig. 4D, 4E), consistent with Nanog expression at these stages in vivo [23, 24]. Furthermore, PCR analysis showed that all tissues of Nanog promoter-EGFP mice contained the Nanog promoter-EGFP fragments, but they were not found in tails of KM mice used as negative controls (Fig. 4F). Microsatellite analysis of the tails and placenta using D12Mit233 primers confirmed that the transgenic mice were derived from ES cells but placenta was derived from both ES cells and host KM embryos (Fig. 4G).
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Figure 4. Transgenic pups produced by injection of Nanog promoter-EGFP ES cells into eight- or four-cell recipient embryos. (A): Nanog promoter-EGFP plasmid. (B): Newborn pups from tetraploid blastocyst injection were abnormal (left two were overgrowth, right one had intestines outside the body). (C): Nanog promoter-EGFP ES cell mice and two litters (left, 1 week old; right, 3 weeks old) of agouti pups showing germline transmission from ES cells injected into four-cell embryos. Embryos collected from female albino KM mice mated with male Nanog promoter-EGFP ES cell mice showed fluorescence at 72 h (D) from four-cell embryo injection and at 96 h (E) from eight-cell embryo injection. (F): Polymerase chain reaction analysis showed Nanog promoter-EGFP fragments in various tissues of Nanog promoter-EGFP ES cell mice. (G): Microsatellite analysis of tails and placenta from three Nanog promoter-EGFP ES cell mice. Abbreviations: EGFP, enhanced green fluorescent protein; GFP, green fluorescence protein; Kan, kanamycin; KM, Kunming; Neo, neomycin; TL, transmitted light.
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Table 3. Efficiency in the production of ES cell mice from Nanog promoter-EGFP ES cells (passage 13) injected into eight- or four-cell embryos or tetraploid blastocysts
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DISCUSSION
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Using piezo drilling and in vitro cultured four- or eight-cell embryos as hosts, we produced viable, healthy, and full germline-competent mice from normal and gene-manipulated mouse ES cells. Compared with the laser-assisted method for producing ES cell mice [16], our method uses a piezo micromanipulator to breach the ZP and directly inject cells under the ZP, thus reducing the time for micromanipulation and lowering the cost by obviating the need for laser equipment. We also used embryos cultured in vitro, allowing flexibility in choosing precise stages of embryos for injection. It was easier and more convenient to obtain eight-cell embryos by in vitro culture in KSOMAA, compared with flushing eight-cell embryos developed in vivo from oviducts, but in vivo-derived embryos may have higher quality and thus better survival after injection [16]. Also, the genetic background of host embryos may affect the overall birth rates and the efficiency of producing ES cell mice. In contrast to the piezo drilling method, the laser-assisted injection method also requires bevelled pipettes, which entail much time and expertise to fabricate [16]. No bevelled pipettes are needed for piezo drilling. The piezo method also has been successfully used for blastocyst injection [22] and routinely used for somatic cell cloning [18]. However, we recognize the toxicity of mercury used for the piezo injection method such that it is not acceptable in human clinics. Thus, some embryologists already tried to replace mercury with Fluorinert FC-77 and obtained satisfactory results after intracytoplasmic sperm injection. However, use of this solution for the piezo method may require more practice and optimization of its loading amount. The problems with mercury for piezo application may eventually be solved by trials with appropriate solutions.
In most injected embryos, ES cells were committed and expanded to the inner cell mass and fetus after culture in vitro and transfer into recipient mice. But in some embryos, ES cells were reduced in number. Previously, single ES cells injected into eight-cell embryos could generate chimeras [25]. Also, at most three ES cells could contribute to the somatic lineages of chimeric mice or produce ES cell mice by tetraploid embryo complementation [26]. Injection of fewer than six ES cells often results in chimeric mice by the eight-cell and laser-assisted methods [16]. More than nine cells have no added benefit because the excessive cells tend to leak out through the opening in the ZP. In initial experiments, we also found no beneficial effects by injecting more [15–20] ES cells. Full-term development of ES cell pups was obtained consistently even when some ES cells were not viable after piezo injection and in vitro culture. The reduction in the number of ES cells could result from disruption of ES cell integrity or quality after injection and culture in KSOM, and this may lead to the birth of chimeras or host-derived mice in some cases. We found that more chimeras were produced when four-cell embryos served as hosts versus when eight-cell embryos served as hosts. Before embryos reach the eight-cell compacted stage, injected ES cells stayed in four-cell embryos for nearly 12 h. The KSOMAA medium and the niche inside four-cell embryos might not be optimal for maintaining the ES cells in a viable and undifferentiated state. This may also explain why more cells from placenta were derived from ES cells in the four-cell embryo injection method. On the other hand, it is possible that damaged ES cells might leak their cytoplasm and thereby "poison" the host embryo. This may lead to death of some ES cell fetuses showing black eyes in both normal ES and Nanog promoter ES cell injection experiments. We anticipate that the efficiency for producing ES cell pups may be increased by instant transfer of injected embryos without culture in vitro or by using appropriate culture medium optimized for survival of both ES cells and embryos.
Using an ES cell line stably expressing the Nanog promoter-EGFP and tetraploid blastocyst injection method, we obtained 10 newborns with black eyes by cesarean section on E19.5. Yet only one newborn survived for 1 week; the others died immediately because of abnormal morphogenesis, including excess weight and exposure of viscera, as reported previously [27]. After Nanog promoter-EGFP ES cells were injected into eight- and four-cell embryos, live mice were obtained. All were derived from Nanog promoter-EGFP ES cells and had full germline transmission. It is likely that placental development from four- or eight-cell embryos better supports the development of transgenic fetuses than does that of tetraploid embryos. It is also possible that random integration of transgenes may disrupt embryo development, which can be partially rescued by four- to eight-cell embryos. Tetraploid blastocysts would not be expected to rescue such minor defects, because development of tetraploid embryos themselves is not normal [28]. We speculate that blastomeres of eight- or four-cell embryos may provide factors that help ES cells overcome defects and developmental blocks, although the underlying mechanisms remain to be determined.
An earlier study suggested that injection into earlier (eight-cell) embryos before the formation of the inner cell mass might result in a higher contribution of ES cells to the chimera [15]. A recent study further demonstrated that injection into eight-cell embryos by the laser-assisted method efficiently yields F0 generation mice that are fully ES cell-derived [16]. To produce ES cell-derived pups, we further extended those studies by piezo-assisted injection of ES cells into an even earlier stage (four-cell embryos) and compared the results with those for eight-cell embryos. We show that use of eight-cell or even earlier stage (four-cell) embryos and the piezo injection method also effectively produced F0 ES cell pups. The four-cell embryos have an advantage over eight-cell stage embryos, because ES cells can be readily deposited into the larger space between the four-cell blastomeres during injection.
Taken together, these results show that ES cell mice can be effectively produced from injection of hybrid ES cells into eight- or four-cell embryos by piezo micromanipulation. The piezo manipulation method can be used as a complementary alternative to the recently published laser-based method [16]. The eight- or four-cell embryo and piezo injection method may better support survival of transgenic embryos with random gene integration. Considering that gene targeting studies usually require a pure genetic background to exclude irrelevant effects on the phenotype, Poueymirou et al. [16] used several inbred ES cell lines (C57, BALB/C, and 129) and obtained efficiency in generating F0 ES pups similar to that with hybrid ES cell lines. The four- or eight-cell embryo and piezo injection method may also be applicable for producing F0 ESC pups from inbred ES cells, but this hypothesis requires further experimentation. It is worth noting, however, that some laboratories actually have used hybrid ES cells to produce gene targeting mice [14]. Furthermore, chimera production by blastocyst injection has been used routinely for testing pluripotency of ES cells or new types of ES-like cells including induced pluripotent stem cells [29–32]. Thus far, development of ES-like cells injected into tetraploid blastocysts represents the most stringent test for developmental potency, because the resulting embryos are composed only of the injected donor cells [31, 32]. The eight- or four-cell embryo and piezo injection method reported here also can be effectively used as the most rigorous test for the developmental potency of new ES or other types of pluripotent stem cell lines.
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
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The authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS
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This study was supported by the Science and Technology Division of Guangdong Province and Guangzhou City, National Nature Science Foundation of China, James and Esther King Biomedical Research Program, and China Scholarship Council.
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FOOTNOTES
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Author contributions: J.H.: experiment execution, collection and/or assembly of data, data analysis and interpretation, manuscript writing; K.D.: experiment execution, provision of study material, collection and/or assembly of data, data analysis and interpretation; H.W.: experiment execution, collection and/or assembly of data; Z.L., Z.C., S.C.: provision of study material, collection and/or assembly of data; L.Z.: administrative support; X.Y.: collection and/or assembly of data; D.L.K.: financial support, final approval of manuscript; L.L.: conception and design, financial support, manuscript writing, final approval of manuscript.
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Abstract
Introduction
