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Regular Article |
a Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia 30912
b Cryobiology Research Institute, Wells Research Center, Indiana University Medical School, Indianapolis, Indiana 46202
ABSTRACT
A series of experiments was conducted to test the hypothesis that an improved cryopreservation protocol for pronuclear stage mouse embryos will produce transgenic (Tg) mice by pronuclear gene injection at a rate not significantly different from noncryopreserved embryos. In the first experiment, three cryoprotective agents (CPAs) (dimethyl sulfoxide [DMSO], propylene glycol [PG], ethylene glycol [EG]) and two cryopreservation protocols, currently used for pronuclear embryos, were compared in regard to their ability to maintain post-thaw morphological integrity and in vitro developmental competence. In the second and third experiments, the optimal cryopreservation protocol determined from the first experiment was used to evaluate in vitro developmental competence of pronuclear embryos following green fluorescence protein gene injection and in vivo developmental competence as well as the gene integration rates. Survival (morphological integrity and development to two cells) of embryos cryopreserved in the presence of DMSO was higher (P < 0.05) than those cryopreserved with either PG or EG. Postinjection developmental competence (development to two cells) of cryopreserved CBA, C57B6/JxCBA-F1 and noncryopreserved (control) embryos was not different (P > 0.05). Postinjection blastocyst formation rate of cryopreserved and noncryopreserved C57B6/JxCBA-F1 embryos was similar (P > 0.05); however, noncryopreserved CBA embryos resulted in a higher blastocyst formation than controls (P < 0.05). While there was no difference in the percentage of transgenic fetuses between cryopreserved and control CBA embryos (P > 0.05), cryopreserved C57B6/JxCBA-F1 embryos resulted in lower transgenic fetuses than control (P < 0.05). These results indicate that the use of cryopreserved mouse pronuclear embryos can be a useful and efficient approach to the production of Tg mice.
cryopreservation, embryo, transgenic
INTRODUCTION
The biotechnology of gene transfer has revolutionized the understanding of biological processes [1]. Production of transgenic (Tg) mice has become routine in many laboratories and large amounts of resources are devoted to the generation of genetically engineered mice (GEM). New lines including inbred, congenic, mutant, Tg and knockout (KO) lines are being developed as models of health and diseases at an exponential rate [2, 3]. To date, pronuclear microinjection of DNA is the most efficient and commonly used means to produce Tg mice [4]. There are, however, several significant constraints regarding the efficient and successful production of Tg animals that include 1) integration of the gene construct, 2) expression of gene constructs in the tissue of choice, and 3) propagation of these Tg animals [5]. Genetic background has been reported as one of the major factors affecting post-thaw survival of mouse embryos [6–10] as well as the generation of Tg mice.
Cryopreservation and low temperature storage of mouse embryos is a cost-effective approach for the maintenance of scientifically important stocks, strains, and lines [11]. The use of cryopreserved mouse pronuclear embryos would provide a more efficient approach to the production of Tg mice using pronuclear injection by reducing or eliminating the need for on-site maintenance of animals to serve as embryo donors and thereby reduce costs associated with animal housing [12, 13].
However, one-cell embryos have fundamental cryobiological characteristics that differ from multicellular embryos [14]. These differences require that cryopreservation procedures used for one-cell embryos must be different than those used for later stage embryos [15]. Increasing cryosurvival of pronuclear mouse embryos would enhance the efficiencies of creating Tg models using cryopreserved embryos. This study was developed to test the hypothesis that optimization of cryopreservation protocols for mouse pronuclear embryos will enable a high Tg rate that is not significantly different from that achieved using noncryopreserved embryos.
MATERIALS AND METHODS
Embryo Collection
CBA and C57BL/6JxCBA-F1 mice were superovulated by an i.p. injection of 5 IU eCG (Sigma Chemical Co., St. Louis, MO) followed 48 h later by 5 IU hCG (Sigma). Females were placed with males immediately after hCG administration, and presumptive pronuclear embryos were collected from the oviducts approximately 16 h after hCG injection. Cumulus oocyte complexes were released into FHM medium (Cell & Molecular Technologies, Inc., Specialty Media Division, Lavallette, NJ) containing 176 U/ml hyaluronidase to remove the cumulus cells. The denuded embryos were washed three times with FHM and three times with KSOM (Specialty Media) medium before they were transferred into a 10-µl drop of KSOM under mineral oil. For each experiment, embryos were collected from each strain, pooled, and divided into two groups. Half of them were cultured in vitro, with or without injection; the remaining half were cryopreserved and used for thawing and injection experiments.
Cryopreservation Procedures
The cryopreservation procedures used were modifications of previously published reports [16–20]. The reason different cooling and warming rates were used between the propylene glycol (PG) and ethylene glycol (EG) groups and dimethyl sulfoxide (DMSO) group was to compare fundamentally different standard methods previously used for mouse oocytes and embryos. These cryopreservation procedures were evaluated using the cryoprotective agents (CPAs) in combination with different cooling and warming procedures. In the first two treatment groups, embryos were initially equilibrated in Dulbecco PBS (D-PBS) containing either 1.0 M PG or EG with 10% fetal bovine serum (FBS) and 0.2 M sucrose in two steps (10-min intervals) at 22°C. Embryos were loaded into 0.25-ml straws (IVM, L'Aigle, France). The straws were then placed into a programmable freezer (Cryologic, PTY Ltd., Melbourne, Australia), cooled at 2°C/min to -6°C when ice nucleation was induced (seeded) manually using cooled forceps. The samples were held at -6°C for 10 min and then cooled at 0.3°C/min to -35°C. After a 10-min holding period at -35°C, the straws were plunged directly into liquid nitrogen (LN2) and stored for 2–6 mo. For thawing, the straws containing embryos were held in air for 15 sec, then placed into a 22°C water bath for 30 sec. The contents of the straws were expelled into sterile petri dishes, and a four-step procedure was performed at 22°C to remove the CPAs by decreasing the concentration of cryoprotectant in 0.25 M steps at 5-min intervals.
Embryos in the third treatment group were loaded into 0.25-ml straws (IVM) containing 10% FBS + 1.0 M DMSO + 0.2 M sucrose in D-PBS that were cooled to 4°C before loading. Then, the straws containing embryos were held at 4°C for 30 min before being transferred to a programmable freezer. The embryos were cooled at 2°C/min to -6°C. Ice nucleation (seeding) was induced by precooled forceps, and cooling was continued at 0.5°C/min to -80°C. The straws were then plunged into LN2 and stored for 2–6 mo. The embryos were thawed by placing the straws into a programmable freezer at -80°C, then warming them at 8°C/min to 4°C. The samples were held at this temperature for 10 min before being transferred into FHM medium at 22°C. Embryos were washed three times, then transferred into 10 µl of KSOM under mineral oil.
Plasmid Construction
A plasmid vector (pACTIN-EGFP) was constructed in plasmid pEGFP-1 (Clontech Laboratories, Palo Alto, CA) by cloning the ß-actin gene promoter into a XhoI/Sall site of the pEGFP-1 so that the Gfp gene in this construct is expressed under the control of the ß-actin gene promoter from mice. pACTIN-EGFP was then transformed into competent Escherichia coli (DH5
) cells. Transformed cells were grown and used to purify the plasmid DNA using the Qiagen maxi preparation kit (Qiagen Inc., Valencia, CA) according to the manufacturer's protocols. Purified plasmid DNA was quantitated by spectrophotometric analysis, aliquoted into small volumes, and stored at -20°C. Plasmid DNA before injection was digested with restriction enzymes XhoI and AflII and a 2.2-kilobase (kb) DNA fragment corresponding to ß-actin-Gfp was separated by electrophoresis on a 1% agarose gel and purified from the gel using the Qiagen gel purification kit. Purified DNA was quantitated and diluted to 4 ng/ml in Tris-EDTA buffer at pH 3.8. Ten-microliter aliquots of DNA were prepared and stored at -20°C until injection. In addition to embryos injected with Gfp, some embryos were cultured without injection (control embryos), while others were injected with a cytomegalovirus-glutathione synthetase construct (positive control) as described previously [21].
Gene Injection and In Vitro Embryo Culture
Cryopreserved embryos were incubated in KSOM for 1 h before pronuclear gene injection. Embryos with two pronuclei were selected under a stereomicroscope and transferred to a 20-µl microdrop of FHM medium [22] under mineral oil. Selected embryos for manipulation were transferred to the injection chamber. Approximately 2 pl of DNA solution (2 ng/pl) were microinjected into the male pronuclei. After microinjection, embryos were washed three times with FHM medium, and 20 embryos were placed into 10-µl drops of KSOM at 37°C in a humidified atmosphere of 5% CO2 and balanced with N2. Developmental stages were observed and medium was renewed (100% of the volume was changed) every 48 h. Embryo culture was continued to 96 h post-thawing to allow viable embryos to reach the blastocyst stage. Embryos that did not continue to advance were removed at the time of media change.
Embryo Transfer
After 24 h of in vitro culture, embryos were transferred into the oviducts of 0.5-day pseudopregnant ICR females. The recipient animals were killed 10 days following embryo transfer to evaluate both implantation rate and expression of EGFP in the resulting fetuses.
Statistical Analyses
Statistical analysis was performed using Sigma Stat Software Program (Version 1.0, Jandel Corporation, San Rafael, CA). Data from the different cryoprotectant treatments were compared with 
2 analysis using a Yates correction for continuity. Standard ANOVA was used to analyze differences in the numbers of live embryos among experimental groups. Data were transformed using an arcsine transformation. The Bonferroni t-test was used to determine differences among groups [23].
RESULTS
Comparison of Cryopreservation Protocols for Mouse One-Cell Embryo
In experiment 1, three different cryopreservation protocols were compared for pronuclear stage mouse embryos. Cryosurvival was assessed by 1) an intact zona pellucida and plasma membrane post-thaw and 2) in vitro developmental competence to the blastocyst stage. Cryosurvival rates of embryos derived from CBA and C57B6/JxCBA-F1 mice are shown in Table 1. Initial post-thaw survival rates of embryos cryopreserved in 1 M DMSO + 0.2 M sucrose (DMSO) were higher (76.7 ± 14.0% and 89.0 ± 9.9%; mean ± SEM) (P < 0.05) than those for embryos cryopreserved in either 1 M PG + 0.2 M sucrose (PG) (48.2 ± 12.8 and 58.7 ± 13.4) or 1 M EG + 0.2 M sucrose (EG) (41.0 ± 10.4 and 46.0 ± 17.1%) for CBA and C57B6/JxCBA-F1, respectively. There was also a significant effect of CPA on blastocyst formation rate. Embryos cryopreserved in DMSO developed to blastocyts at higher (P < 0.05) rates (68.9 ± 7.9 and 89.1 ± 10.1 for CBA and C57B6/JxCBA-F1, respectively) than those cryopreserved in either PG (46.3 ± 7.6 and 67.2 ± 6.0%, respectively) or EG (51.8 ± 2.1 and 66.8 ± 3.0%, respectively).
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Effects of Cryopreservation and Gene Injection on the Survival of Embryos
In experiment 2, the effect of pronuclear injection on CBA and C57B6/JxCBA-F1 embryos cryopreserved in the presence of DMSO was investigated; the results are presented in Table 2. There were no significant differences (P > 0.05) in initial post-thaw survival (84.7 ± 9.5% and 90.2 ± 7.5%) or development to the two-cell stage (83.7 ± 5.2 and 94.6 ± 4.2%) for CBA and C57B6/JxCBA-F1 embryos, respectively. However, while the percentage of cryopreserved CBA embryos that developed to the blastocyst stage (62.8 ± 8.5%) was significantly lower (P < 0.05) when compared to noncryopreserved CBA embryos (80 ± 5.4%), there was no significant difference in the endpoint (P > 0.05) between cryopreserved (86.1 ± 3.7%) and noncryopreserved (93.5 ± 2.1%) C57B6/JxCBA-F1 embryos.
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Efficiency of Production of Tg Mice Using Cryopreserved Embryos
The results of experiment 3 are shown in Table 3 and show the implantation and Tg rates of noncryopreserved and cryopreserved CBA and C57B6/JxCBA-F1 embryos following gene injection. Implantation (32.8 ± 6.3% versus 45 ± 3.4%) and Tg rates (3.7 ± 1.5% versus 4.3 ± 3.1%) of noncryopreserved and gene-injected CBA embryos were not different from those cryopreserved counterparts (P > 0.05). Although implantation rates of noncryopreserved (47.3 ± 3.1%) and gene-injected C57B6/JxCBA-F1 embryos were not different from those cryopreserved (38.6 ± 6.7%) counterparts (P > 0.05), the percentage of Tg fetuses was significantly higher (P < 0.05) for noncryopreserved (8.0 ± 2.0%) than those cryopreserved counterparts (5.9 ± 3.0%).
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DISCUSSION
In the present study, we evaluated the cryopreservation protocols for pronuclear stage mouse embryos that would yield acceptable rates of Tg mice when used with pronuclear injection. During cell cryopreservation, typically, there is a strong relationship between the initial slow cooling rate and the temperature at which slow cooling is terminated. If the temperature at which the sample is plunged into liquid N2 (-196°C) is high (e.g., 
-35°C), cells may not have enough time to dehydrate sufficiently and will therefore undergo intracellular ice formation (IIF), which is most often lethal. However, if the temperature at which the sample is plunged into liquid N2 is low (e.g., 
-80°C), the risk of IIF will be low because slow cooling (0.3–0.5°C/min) to those low temperatures usually provides sufficient time for intracellular water to leave the cell via exosmosis. In this regard, oocytes and one-cell embryos have certain disadvantages compared to cleavage-stage embryos, including their large initial water volume and their low surface area-to-volume ratio that predisposes them to IIF if the initial slow cooling rate and/or the plunging temperature is relatively high [16, 24]. Although several standard protocols are very effective in cryopreserving cleavage-stage mouse embryos, when similar protocols are used for one-cell embryos, they often result in low survival.
Using fundamental cryobiology parameters, a previous study calculated the optimal combination of freezing protocol parameters for metaphase II mouse oocytes in the presence of DMSO. These were predicted to be a cooling rate of 0.59°C/min with a plunging temperature of below -67°C, and this prediction has been experimentally validated [16]. Furthermore, our group previously determined [14] that there was no significant difference between mouse oocytes and one-cell mouse embryos in regard to plasma membrane hydraulic conductivity (Lp: 0.77 versus 0.81 µm/min/atm) and DMSO permeability (PDMSO: 1.8504 x 10-3 versus 2.04 x 10-3 cm/min) coefficients. This suggests that these two cell types show a similar osmotic response, and thus the appropriate cooling and warming rates and plunging temperature predicted for oocytes should be suitable for one-cell mouse embryos.
In the present study, slow cooling (0.3°C/min) in the PG and EG groups was terminated at -35°C, while in the DMSO group, slow cooling (0.5°C/min) was terminated at -80°C. In addition, while rapid warming (
250°C/min) was used in the PG and EG groups, embryos in the DMSO group were warmed slowly (8°C/min). The reason different cooling and warming rates were used between the PG and EG groups and the DMSO group was to compare fundamentally different standard methods previously used for mouse oocytes and embryos. The DMSO treatment group parameters in this study were based upon recent improvements in procedures leading to increased survival of cryopreserved mouse metaphase II oocytes using both theoretical [16] and empirical approaches [18, 20]. The approaches developed in these reports used 1.5–3 M DMSO, an initial slow cooling rate of 0.5°C/min, a plunging temperature of -80°C, and a warming rate of (8°C/min). In the current study we used a similar procedure but reduced the DMSO concentration to 1 M and supplemented the freezing solution with 0.2 M sucrose and 10% FBS. This modification was made for the following reasons. It has been demonstrated that addition of nonpermeating compounds (e.g., sucrose) to the freezing solution protects mammalian cells by dehydration via the increase in extracellular osmolality. This subsequently decreases the risk of lethal IIF during freezing and decreases osmotic stress by acting as an osmotic buffer during the CPA removal [15, 17, 25, 26]. Additionally, the use of some macromolecules such as BSA, fetal calf serum (FCS), and FBS has been reported to increase the post-thaw fertilization rate of mouse oocytes [18]. Using these procedures, higher survival rates were obtained in the current study when embryos were cooled to -80°C (at 0.5°C/min) and warmed to 4°C (at 8°C/min) in the presence of 1 M DMSO + 0.2 M sucrose + 10% FBS. These results are consistent with previous studies demonstrating that mouse oocytes [16, 20] and one-cell [17] and two-cell [27] embryos that were slowly cooled to -80°C, require slow (8°C/min) rather than rapid warming.
It has previously been demonstrated that mouse cleavage-stage embryos from different genetic backgrounds respond differently to a standard cryopreservation procedure [6, 8]. In this study, post-thaw developmental competence of pronuclear embryos derived from the inbred strain (CBA) was significantly lower than those embryos derived from a hybrid strain (C57B6/JxCBA-F1). Therefore, the current study further demonstrates the existence of the difference in cryosurvival between hybrid and inbred embryos at the pronuclear stage.
To utilize cryopreserved mouse embryos in the production of Tg animals, it is essential that they withstand multiple potentially damaging events inherent in the cryopreservation and micromanipulation procedures. Previously, Leibo et al. [12] injected various gene constructs into frozen-thawed, hybrid (ICR x FVB) embryos that were slowly cooled (0.8°C/min) to -70°C in 3 M EG and rapidly thawed (250°C/min) to room temperature. In that report, 65% of frozen-thawed pronuclear mouse embryos survived microinjection. Later, Tada et al. [13] vitrified hybrid mouse embryos (C57BL/6N x C3H/HeN) in solution containing 2.75 M DMSO + 2.75 M PG + 1 M sucrose. In that study, while 75% of the cryopreserved embryos were suitable for microinjection post-thaw, 68% (of the 75%) of the of embryos survived microinjection. In the current study, to determine the microinjection efficiencies with cryopreserved embryos (frozen in the presence of 1 M DMSO + 0.2 M sucrose), further experiments were performed by post-thaw gene injection. High postinjection development (to the two-cell stage) was achieved following pronuclear injection of cryopreserved CBA (83.7%) and C57B6/JxCBA-F1 (94.6%) embryos. Therefore, the current results clearly indicate that a significant percentage of the cryopreserved embryos can be utilized for microinjection if the embryos are cryopreserved with the procedure determined here. Although embryos derived from hybrid and outbred (e.g., ICR) mice typically demonstrate high survival after cryopreservation, there is growing information which suggests that the developmental competence of embryos derived from inbred (e.g., CBA, FVB, BALB/c) mice is reduced after cryopreservation compared to nonfrozen embryos from the same strains [9]. Thus, the higher development competence of C57B6/JxCBA-F1 embryos in the present study may be explained, at least in part, by hybrid robustness.
To determine the Tg efficiency, cryopreserved and nonfrozen control embryos were injected with a gene construct designed to be expressed during early mouse development. In vivo developmental competence and the gene integration rates of embryos were determined by transferring these embryos to recipient animals. The results of the current study show that implantation and transgenesis rates of noncryopreserved and cryopreserved embryos are similar for CBA embryos. However, noncryopreserved C57B6/JxCBA-F1 embryos yielded a higher rate of Tg mice production than cryopreserved embryos. Furthermore, the percentage of Tg mice obtained from C57B6/JxCBA-F1 embryos was significantly higher (P < 0.05) than those of CBA embryos. These data are in accordance with the observation made by Brinster et al. [5] where C57/BL6xSJL hybrid mice resulted in higher Tg rates than C57/BL6 inbred mice.
The present study demonstrates that multiple interventions such as cryopreservation and micromanipulation can be performed without significant loss of embryo development and transgene expression. Table 4 compares overall Tg efficiencies of past and the current studies. Compared to previous reports [12, 13], a higher overall Tg rate was obtained in the present study. These data suggest that with the injection of 300 noncryopreserved embryos, it is possible to obtain approximately 13 and 24 Tg mice using inbred (CBA) and hybrid (C57B6/JxCBA-F1) strains, respectively. Similarly, with the storage of 300 embryos from CBA and C57B6/JxCBA-F1 strains, it would be possible to produce about 11 and 18 Tg mice, respectively. Production of such numbers of Tg mice would be sufficient to allow establishment of a Tg line and colony expansion by natural mating.
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In summary, blastocyst development rates of embryos from either genetic background were highest using DMSO. C57B6/JxCBA-F1 embryos survived cryopreservation at a higher frequency than CBA embryos. When embryos were microinjected following cryopreservation, postinjection development to the two-cell stage was not different for CBA and C57B6/JxCBA-F1 embryos, but CBA embryos, after injection, had a reduced development to the blastocyst stage. Overall, cryopreserved and noncryopreserved CBA embryos provided Tg fetus rates that were not significantly different. However, there was a cryopreservation treatment by genotype interaction in which cryopreserved C57B6/JxCBA-F1 embryos resulted in a significant reduction of Tg fetuses relative to noncryopreserved controls. These results support the hypothesis that optimized cryopreservation procedures can result in Tg efficiencies that are not different between cryopreserved and noncryopreserved mouse embryos. Further investigation of the observed cryopreservation by genotype interaction is currently being conducted. These data demonstrate that cryopreservation of pronuclear-stage mice embryos for Tg production can be an efficient way to manage mouse colonies and may have important application in the area of Tg research involving pronuclear injection by solving some of the logistic problems related to producing Tg mice.
FOOTNOTES
First decision: 17 October 2000.
1 Correspondence: John K. Critser, Cryobiology Research Institute, The
Herman B. Wells Center for Pediatric Research, Riley Hospital for Children,
1044 West Walnut Street, Indianapolis, IN 46202. FAX: 317 274 8679; jcritser{at}iupui.edu 
Accepted: March 26, 2001.
Received: August 24, 2000.
REFERENCES
H, McGraw RA, Brackett BG. Production and identification of transgenic mice for cytomegalovirus-controlled expression of glutathione synthetase. In: Southeastern Chapter of the Society of Toxicology Annual Meeting (SESOT); 2–3 October 1997; Athens, GA
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