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Reproduction (2005) 130 53-59
DOI: 10.1530/rep.1.00667
Copyright © 2005 Society for Reproduction and Fertility
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RESEARCH

The genetic heterozygosity and fitness of tetraploid embryos and embryonic stem cells are crucial parameters influencing survival of mice derived from embryonic stem cells by tetraploid embryo aggregation

Xiangyun Li1, Wei Wei2, Jun Yong3, Qing Jia1, Yuansong Yu2 and Keqian Di1

1 College of Animal Science and Technology, Agricultural University of Hebei, Baoding, Hebei 071001, China, 2 College of Life Science, Peking University, Beijing 100871, China and 3 Beijing Vitalriver Laboratory Animal Inc, Beijing 100012, China

Correspondence should be addressed to X Li; Email: lixiangyun35{at}yahoo.com.cn


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The aim of this paper was to determine whether the genetic background of tetraploid embryos contributed to the survival of mice derived from embryonic stem (ES) cells by tetraploid embryo complementation. Twenty-five newborns were produced by aggregation of hybrid ES cells and tetraploid embryos with different genetic backgrounds. These newborns were entirely derived from ES cells judged by microsatellite DNA (A specific sequence of DNA bases or nucleotides that contains mono, di, tri or tetra repeats) and coat colour phenotype and germline transmission. Fifteen survived to adulthood while seven died of respiratory failure. All newborns were derived from outbred or hybrid tetraploid aggregates and no newborns were from the inbreds. Our results demonstrate that the genetic heterozygosity, fitness of tetraploid embryos and fitness of ES cells are crucial parameters influencing survival of mice derived from ES cells by tetraploid embryo aggregation. In addition, this method represents a simple and efficient procedure for immediate generation of targeted mouse mutants from genetically modified ES cell clones, in contrast to the standard protocol, which involves the production of chimeras and several breeding steps.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Embryonic stem (ES) cells, derived from preimplantation embryos, are undifferentiated, immortal cells capable of differentiating into derivatives of all three embryonic germ layers. Pluripotent ES cells have been used extensively in studies of embryogenesis, gene function and development in the mouse. Tetraploid mouse embryos are not capable of completing normal development independently (Kaufman & Webb 1990, Eakin & Behringer 2003), but when they are complemented by the introduction of ES cells, they can then develop into normal conceptuses derived completely from ES cells (Ueda et al. 1995, Nagy et al. 1990, 1993, Wang et al. 1997, Eggan et al. 2001, 2002, Schwenk et al. 2003, Wang & Jaenisch 2004).

Mice derived completely from ES cells by tetraploid embryo complementation (Eggan et al. 2001, 2002) are termed ES mice. The first production of ES mice appeared in the year of 1990 (Nagy et al. 1990). Unfortunately, these ES mice died of respiratory failure and no mice survived to adulthood. With development some adult ES mice were generated from R1 ES cells derived from an intercross between two different 129 substrains (129/Sv and 129/Sv-CP; Nagy et al. 1993). The genetic heterozygosity of ES cells is a crucial parameter influencing post-natal survival of ES mice and adult ES mice can be generated more successfully from hybrid ES cells in culture or after consecutive rounds of drug selection (Eggan et al. 2001, 2002, Schwenk et al. 2003). In contrast to cloned mice, ES mice have smaller birth and placental weights (Eggan et al. 2001) and show apparently normal morphological, physiological and neurological characteristics (Schwenk et al. 2003). In addition, the methods of generating ES mice are much more simple and effective, this can present us with a shortcut to study gene function. Cloning from somatic cells is inefficient, however, the cloning efficiency can be raised substantially when tetraploid embryo complementation is applied to the cloning procedures (Eggan et al. 2004, Hochedlinger & Jaenisch 2002).

The tetraploid components of reported ES mice were derived from CD1, B6CBAF2, or B6D2F2 (Nagy et al. 1990, 1993, Wang et al. 1997, Eggan et al. 2001, 2002). The genetic background differences in the developmental potential of tetraploid embryos have been suggested in some results (Kaufman & Webb 1990, Henery & Kaufman 1991, Kaufman 1991a, 1991b, Eakin & Behringer 2003), however, the issue has not been substantially explored in the currently available literature on ES mice.

By aggregation of hybrid ES cells with tetraploid embryos derived from different strains, we compared the genetic background differences of tetraploid embryos in the generation of ES mice and detected the tetraploid contribution in adult ES mice by a microsatellite DNA marker instead of the glucose phosphate isomerase (GPI) marker in this study.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Mice
Inbred (129/Sv, C57BL/6, BALB/c, DBA/2), outbred (CD1) and hybrid (CD1 x 129/Sv, C57BL/6 x 129/Sv, BALB/c x 129/Sv, B6D2F1 x 129/Sv, B6D2F1 x CD1, B6D2F1, B6D2F2).

All mouse stocks purchased from Beijing Vitalriver Laboratory Animal Inc. Beijing, China were maintained on a constant light-dark cycle (19:00–07:00 dark, 07:00–19:00 light). Animals where provided with commercial pelleted food and water ad libetum. All experimental protocols and animal handling procedures were reviewed and approved by the Animal Care and Use Committee of Peking University.

Embryo culture
All embryo culture was carried out in microdrops on standard bacterial Petri dishes (Nunc Roskilde, Denmark) under mineral oil (Sigma). KSOM media (potassium simplex optimized medium; Nagy et al. 2003) was used for embryo culture. M2 media (Sigma; Nagy et al. 2003) was used for room temperature operations whereas long-term culture was carried out in bicarbonate-buffered KSOM at 37.5 °C with an atmosphere of 5% CO2 in air.

Isolation of Heb1 ES cells
We derived our own hybrid ES line instead of testing a commonly used ES cell line to generate ES mice. The reason for this is that hybrid ES cells have been previously reported to give good postnatal survival (Eggan et al. 2001, 2002, Schwenk et al. 2003, Wang & Jaenisch 2004, Li et al. 2005). In this study, Heb1 hybrid ES cells aggregated with tetraploid embryos was isolated from inbred C57BL/6 females (three to four week old) mated to adult inbred 129/Sv males. In brief, blastocysts were collected 3.5 days postcoitum from C57BL/6 females and cultured in ES cell media on a mitomycin C- treated mouse embryonic fibroblasts as previously described (Nagy et al. 2003); at day 5 the outgrowth was dissociated by pipetting in trypsin solution (Gibco, Grand Island, NY, USA), and the cell suspension was replated on a fresh feeder layer. ES cell media was Dulbecco’s modified Eagle’s medium (DMEM; Gibco) with 20% fetal calf serum (Gibco) containing 1000 units/ml of leukemia inhibitory factor (Gibco). These plates were screened 3 days later for the presence of ES cell colonies. We started counting passage numbers when we were first able to pass the cells into 35 mm plates (passage 1). Three ES cell lines (designated Heb1, Heb2 and Heb3) were established and the Heb1 cell line (less than 10 passages) was aggregated with tetraploid embryos.

Production of tetraploid embryos
The different strains of females (three to four week old) were superovulated (Nagy et al. 2003) and mated with the corresponding males (eight to ten week old). The presence of a vaginal plug the next morning was taken as evidence of mating and this was considered to be the first day of gestation. On the morning of the second day of gestation, the oviducts of females were flushed with M2 media to recover late two-cell embryos. The two-cell embryos were placed between two platinum electrodes laid 1 mm apart in a nonelectrolyte solution containing 0.3 M mannitol, 0.1 mM calcium chloride, 0.1 mM magnesium sulfate, and 0.3% BSA (Sigma) in the electrode chamber (Microslide 450-1, BTX Inc, San Diego, CA, USA). The blastomeres were fused by two short electric pulses (100V for 50 µsec) applied by an Electro Cell Manipulator (ECM2001, BTX Inc.) (Kubiak & Tarkowski 1985).

Aggregation of ES cells and tetraploid embryos
Twenty-four hours after electrofusion, most of the fused tetraploid embryos developed to the four-cell stage. Only these four-cell stage embryos were used for aggregation. The zonae pellucidae of these embryos were removed by treatment with 0.5% pronase (Sigma) solution. ES cell colonies with undifferentiated state were chosen and briefly trypsinized to form clumps of loosely connected cells. Clumps of 20–30 ES cells were then sandwiched between two tetraploid embryos in aggregation wells made by pressing a darning needle into the plastic bottom of the culture plate (Nagy et al. 1993, 2003). The aggregates were cultured overnight in microdrops of KSOM media until transfer to recipient females.

Cell counting of tetraploid blastocysts
Forty-eight hours after electrofusion, tetraploid blastocysts were cultured in KSOM media containing 5 µg/ml Hoechest 33342 (Sigma). After culture for 20 min, the tetraploid embryos were pressed on slides and the cell number of each embryo was counted under an inverted fluorescence microscope. One-way ANOVA was used to determine if there was significant variance above these data, which, if found, was then followed by the Tukey–Kramer multiple comparisons test to perform pairwise comparisons between individual means (InStat, GraphPad Software, San Diego) and the level of significance was set at P < 0.05.

Caesarean section
Aggregates (8 to 10) were transferred into each uterine horn of 2.5 days postcoitum pseudopregnant outbred CD1 females (six to seven week old) that had mated with vasectomized males. Pregnant recipients were routinely subject to a Caesarean section (to collect placentae) on day 18.5 of pregnancy. All fetuses were counted, the live were fostered to lactating hybrid B6D2F1 (C57BL/6 x DBA/2) mothers, aged 8–10 weeks and the weights of fetuses and placentae were recorded. One hour after birth, lung sections of neonatal fetuses that died of respiratory failure were produced and examined. Adult ES mice were mated with outbread CD1 female mice, aged 8–10 weeks, to test for germline transmission. After test breeding, all mice were killed and various tissues were dissected for microsatellite DNA analysis.

PCR analysis of genomic DNA
PCR amplification of the microsatellite DNA marker (Massachusetts Institute of Technology, Cambridge, MA, USA) D5Mit138 (Dietrich et al. 1992) was performed by using primer pairs obtained from another group in our lab (Qing TT, TanL). DNA was extracted from 13 tissues of adult ES mice (including heart, lung, liver, kidney, blood, stomach, spleen, pancreas, brain, muscle, testis, intestine and bladder) and part placentae. Reaction samples (25 µl) were subjected to 34 cycles of 1 min 95 °C, 1 min 55 °C, 2 min 72 °C and products were separated on a 4% agarose gel. The PCR results were visualised by direct UV-transillumination of the agarose gel.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
Developmental potential of tetraploid embryos
To investigate the developmental potential of mouse tetraploid embryos with different genetic backgrounds, twelve mouse populations were used to produce tetraploid embryos (Table 1Go). In the 12 populations, 129/Sv, C57BL/6, BALB/c, DBA/2 were inbred, CD1 was outbred and the remainder were hybrid mice (eg CD1 x 129/Sv mice). Development of tetraploid embryo was shown to the four-cell and blastocyst stages on 24 h and 48 h after electrofusion (Table 1Go). Most of the 2-cell embryos (77.8%–95.7%) were fused by two short electric pulses (100V for 50µs), and the fusion rate was unaffected by genetic backgrounds (P > 0.05) except 129/Sv and C57BL/6 x 129/Sv which exhibited lower fusion rates (77.8% and 79.2%, respectively). A higher proportion of outbred and hybrid tetraploid embryos (77.0%–86.1%) developed to the blastocyst stage by 48 h post electrofusion than for inbred ones (22.2%–28.6%). The mean cell number range of inbred tetraploid blastocysts in the range of 10.4–13.2 cells, hybrid cell number scope was in the range of 17.5–21.1 cells and outbred cell number scope was 19.9. Except for BALB/c x 129/Sv and B6D2F1 x 129/Sv, the cell number scope was not significantly variable between the cell number of outbred and hybrid blastocysts, but there was significantly greater variance than in inbred embryos. In general, this data suggests that outbred and hybrid tetraploid embryos have advanced developmental speed (P < 0.05).


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  		Table 1 Developmental potential of mouse tetraploid embryos in vitro.
 
Survival of ES mice
To test whether the genetic backgrounds of tetraploid embryos were playing a role in the survival and generation of ES mice, three ES cell lines, designated Heb1, Heb2 and Heb3, were established. All ES cell colonies usually have a characteristic morphology as tight round colonies with smooth edges. It was difficult to distinguish the individual cells in these colonies, although their nuclei can be recognized and can contain one or two prominent nucleoli. Since the Heb1 cell line (Fig. 1Go) had a relative fast rate of growth, these ES cells were chosen to aggregate with tetraploid embryos (Fig. 2Go) of different genetic backgrounds. We aggregated 907 tetraploid embryos with the Heb1 cells. After transfer to recipient females, these composite embryos (Fig. 3Go) gave rise to 58 fetuses (Fig. 4Go) that were alive and active at caesarian section. However, fifteen died of respiratory failure within 30 min, four were eaten by their foster parent, two were lost during fostering and three died of unknown reasons on days 3, 7 and 11 after birth. The remaining 34 fetuses developed to adulthood (Table 2Go). Because (C57BL/6 x 129/Sv)F1 hybrid mice are agouti, ES mice derived completely from C57BL/6 x 129/Sv hybrid ES cells were expected to have agouti coats. In the study, all ES mice were male and had agouti coats. The results also indicated that the Heb1 cell line is male. Furthermore, all adult ES mice showed normal morphological characteristics and produced normal agouti offsprings (Fig. 5Go) when mated with albino CD1 mice. Examination of the lungs from fetuses, which died of respiratory failure, revealed that the alveoli were not inflated. In contrast, the lungs of control fetuses were fully inflated and alveoli functional. Fifty-five fetuses were derived from hybrid aggregates, two derived from outbred aggregates, one derived from inbred DBA/2 aggregates and no fetuses were from inbred 129/Sv, C57BL/6 and BALB/c aggregates. B6D2F1 and B6D2F2 and B6D2F1 x 129/Sv tetraploid aggregates had significantly higher success (P < 0.05) in production of ES mice and B6D2F2 and B6D2F1 x CD1 and CD1 x 129/Sv had significantly higher survival rates (P < 0.05) of postnatal ES mice (Table 2Go). These results indicated that the developmental potential of the tetraploid aggregates is strain dependent and furthermore, outbred or hybrid tetraploid embryos have an advantage on the production of ES mice (P < 0.05).



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  		Figure 1 Hybrid Heb1 embryonic stem (ES) cell line and mouse embryonic fibroblasts (MEPs). Magnification x 200.

 


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  		Figure 2 Tetraploid four-cell embryos after removal of zonae pellucidae. Magnification x 200.

 


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  		Figure 3 A blastocyst derived from the aggregation of tetraploid embryos and ES cells. Magnification x 200.

 


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  		Figure 4 Neonatal ES mice produced by aggregation of Heb1 ES cells and B6D2F2 tetraploid embryos.

 

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  		Table 2 Survival of ES mice generated from tetraploid embryo aggregation.
 


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  		Figure 5 The offspings of a male ES mouse (agouti) mated to a CD1 female (albino).

 
Embryonic and placental overgrowth in ES mice
Embryonic and placental overgrowth is a widespread phenomenon in cloned animals (Cibelli et al. 1998, Wakayama et al. 1998, 1999, Wakayama & Yanagimachi 1999, Wells et al. 1999). In this study, 42 newborns and 36 placentae were weighed and found to have a mean birth weight of 1.67 ± 0.41g and a mean placental weight of 0.17 ± 0.08g, the largest birth and placental weights were 2.35g and 0.30g, respectively. These weights were significantly higher than those of control newborns (mean birth weight 1.13 ± 0.06g, mean placental weight 0.12 ± 0.02g, n = 34, P < 0.01). Data from control newborns were recorded from litters with a size of less than or equal to three. The mean birth weights of the 15 ES newborns which died of respiratory failure was 1.90g, that of the unknown cause of death embryos was 1.52g and this weight difference is significant at P < 0.01. This suggests that newborn ES mice with bigger birth weight more easily undergo respiratory failure.

Tetraploid contribution in ES mice
Due to the limited sensitivity and polymorphism of the GPI assays, the phenotype of microsatellite DNA marker D5Mit138 instead of GPI was applied to detect occasional tetraploid contribution in ES mice. Only a small fraction of B6D2F2 tetraploid embryos were not differentiated from Heb1 ES cells at the microsatellite marker. We isolated genomic DNA from 13 tissues of each adult ES mouse derived from B6D2F2 tetraploid-ES aggregates and 12 placentae, subjecting the samples to PCR analysis. The results of PCR showed that only a lung sample and 8 placentae were confirmed to contain tetraploid contribution. No tetraploid contribution was found in other samples (Table 3Go).


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  		Table 3 Microsatellite DNA analysis of tissues from adult ES mice. All DNA samples were isolated from adult ES mice derived from B6D2F2 tetraploid-ES aggregates. Tissues containing DBA/2 component are indicated +.
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
We were interested to determine whether the genetic backgrounds of tetraploid embryos influenced the production and survival rates of ES mice. By aggregation of ES cells with tetraploid embryos of different genetic backgrounds, we have produced 34 adult mice derived entirely from ES cells, as judged by microsatellite DNA, coat colour phenotype determination and germline transmission. Our results indicated that the genetic backgrounds of tetraploid embryos are a crucial parameter influencing production of ES mice.

In this study, viable ES mice where efficiently produced by aggregation of hybrid Heb1 ES cells with hybrid tetraploid embryos. This result is consistent with our proximate reports (Li et al. 2005) and other earlier reports (Eggan et al. 2001, 2002, Schwenk et al. 2003) showing that the genetic heterozygosity of ES cells is a crucial parameter influencing postnatal survival in ES mice.

Using a microsatellite DNA marker, 15 adult ES mice and 12 placentae were detected for tetraploid contributions and only 1 adult ES mouse and 8 placentae were found to contain a tetraploid component. These results are consistent with previous reports (Nagy et al. 1993, James et al. 1995, Wang et al. 1997, Eakin & Behringer 2003, Eggan & Jaenisch 2003) showing that tetraploid cells are frequently present in the extraembryonic lineages. In the development of tetraploid-ES cell aggregates, the proliferation rate of tetraploid cells is sure to influence the embryonic implantation and placental development (Adamson et al. 2002). The outbred and hybrid tetraploid embryos were superior to the inbreds in this factor, as judged by the developmental speed and cell number of blastocysts in in vitro culture. In this study, only a newborn ES mouse (with no adult survival) was derived from DBA/2 tetraploid aggregates and other 57 newborn ES mice and 34 adult ES mice were derived from outbred or hybrid tetraploid aggregates. This data showed that the genetic backgrounds of tetraploid embryos are the other important factors influencing the production of ES mice. Although no significant variances were found in the developmental speed and cell number of the six kinds of tetraploid blastocyst (including CD1, CD1 x 129/Sv, C57BL/6 x 129/Sv, B6D2F1 x CD1, B6D2F1, and B6D2F2), the efficiency (2.8%–14.1%) of generating ES mice from six kinds of aggregates were significantly different to each other(P < 0.01). This suggested that genetic fitness between tetraploid embryos and ES cells also influences the production of ES mice. In addition, while we neglected to keep the number of aggregates transferred per recipient constant for each strain, this is another possible factor affecting the efficiency of producing ES mice – since it is generally accepted that the number of fetuses developing within a pregnant mouse can affect the viability of those fetuses. Among the 12 kinds of tetraploid embryos, B6D2F2 is best source for production of ES mice, as these tetraploid embryos exhibit no 2-cell block, have high fertilization rates, outstanding developmental potential and are readily available from breeders.

The embryonic and placental overgrowth is not unique to ES mice but likely to result from the common experimental procedure in culturing embryos, because a similar weight increase has been found for control mice derived from in vitro-cultured embryos (Eggan et al. 2001). However, the weight increase in ES mice was occasionally severe. This suggested that the number of ES cells and tetraploid cells in aggregates probably contributes to the increased weight. In addition, the ES mice with larger birth weight often suffered respiratory failure. Respiratory failure was a main factor in the premature death of post-natal ES mice. Because respiratory effort only occurs after birth, abnormal development in lung function probably does not lead to pregnancy failure in prenatal ES mice. The respiratory failure phenomenon has also been described in other cloned animals (Cibelli et al. 1998, Wakayama et al. 1998, 1999, Wakayama & Yanagimachi 1999, Wells et al. 1999).

In addition to aggregation, ES mice can also be produced by injection of ES cells directly into tetraploid blastocysts (Wang et al. 1997, Eggan et al. 2001, 2002, Schwenk et al. 2003, Wang & Jaenisch 2004). Although the efficiency of aggregation is slightly lower than that of injection, aggregation does not require the elaborate microinjection apparatus and requires far less practice before proficiency of the technique is achieved. Furthermore, aggregation can produce at 100–150 aggregates per hour vs 20–30 by means of blastocyst injection. In a word, the aggregation appears to be more popular technique than the injection for the production of ES mice.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
We thank Dr Ting Ting Qing and Dr Lei Tan for PCR primer pairs. We also thank Mrs Jie Fang You and Mrs Xiao Ran Xiong for cell culture expertise. We are also grateful to Dr Han Qin and Dr Zan Tong for microsatellite DNA analysis. This work was supported by Grants-in-Aid from Beijing Municipal Science & Technology Commission and the President Foundation from Agricultural University of Hebei. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.


   Footnotes
 
Received 28 January 2005
First decision 1 March 2005
Revised manuscript received 13 April 2005
Accepted 5 May 2005


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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