Cryopreservation of Caprine β-Lactoglobulin Transgenic Mouse Embryos

CRYOBIOLOGY 35, 290–298 (1997)ARTICLE NO. CY972051

Cryopreservation of Caprine b-LactoglobulinTransgenic Mouse Embryos

Elena Ibanez, Mark Grossmann, Francesca Vidal, Josep Egozcue, and Josep SantaloUnitat de Biologia Celrlular, Departament de Biologia Celrlular i Fisiologia,

Universitat Autonoma de Barcelona, 08193 Bellaterra, Spain

As an economical and safer alternative to the maintenance of transgenic animals as live stocks,transgenic embryo cryobanks can be generated and maintained indefinitely. Two-cell embryos obtainedfrom four lines of caprine b-lactoglobulin transgenic mice homozygous for the transgene were cryopre-served, and their response to cryopreservation-related stress was investigated. Significant differencesbetween transgenic lines were found in the viability of frozen/thawed transgenic embryos and also intwo-cell embryo production after superovulatory treatment of transgenic females. The results of thisstudy suggest that cryopreservation protocols should be assessed on each transgenic line before thegeneration of transgenic embryo banks. q 1997 Academic Press

Key Words: cryopreservation; goat b-lactoglobulin; transgenic; mouse embryos.

background to be recovered in the case of ge-The production of transgenic animals hasnetic drift or loss of transgene expression oc-become a routine in many laboratories andcurring in later generations.large amounts of time and money are devoted

Factors such as the site of transgene integra-to the generation of genetically modified ani-tion and the embryo genetic background,mals. Preservation of these valuable animalswhich differ among transgenic lines, may af-is often of prime interest but maintenance offect the efficiency of transgenic embryo cryo-transgenic lines as live stocks is expensive andbanking by disturbing either embryo develop-time consuming.mental potential or embryo sensitivity to cryo-Since 1972, when Whittingham et al. (26)injury. In fact, insertional mutations thatand Wilmut (27) separately described thatimpair embryo development have been de-mouse embryos could be frozen successfullyscribed in several transgenic lines (3, 4, 15),and stored at01967C, the use of cryopreserva-and genotype has been reported as the majortion techniques has rapidly increased world-factor influencing postthaw embryo survivalwide. Frozen stocks of transgenic embryos canin the mouse (7, 19–21). Therefore, embryosbe preserved indefinitely as an alternative tofrom different transgenic lines may differ inthe maintenance of live stocks and offer im-their ability to withstand a standardized cryo-portant advantages. First, transgenic linespreservation protocol.which are not currently in use can be frozen

In a previous work, four lines of transgenicand stored for future studies, thus reducingmice for the caprine b-lactoglobulin (cBLG)costs. Second, routine freezing of transgenicgene were obtained, three of them expressinganimals provide a means to safeguard thesethe exogenous protein in the mammary glandvaluable animals against situations such asat levels ranging from 0.02 to 0.5 mg/ml of

disease, genetic contamination, or breedingmilk (10). With the intention of generating an

failure. Third, freezing transgenic embryos ofembryo cryobank for these transgenic lines, it

early generations allows the original genetic was necessary to assess whether thesetransgenic embryos would be able to recoverand develop normally after cryopreservation-

Received October 21, 1996; accepted August 10, 1997. related stress.

2900011-2240/97 $25.00Copyright q 1997 by Academic PressAll rights of reproduction in any form reserved.

AID CRYO 2051 / a910$$$$$1 10-23-97 16:12:40 cryoas AP: CRYO

291CRYOPRESERVATION OF TRANSGENIC MOUSE EMBRYOS

Thus, the main objective of this study was hybrid F1 female mice (4 to 8 weeks old) weresuperovulated by intraperitoneal injection ofto investigate the postthaw viability of cryo-

preserved embryos from the four lines of 5 IU equine chorionic gonadotrophin (eCG;Foligon, Intervet) followed 48 h later by 5cBLG transgenic mice. To our knowledge, no

specific data about the efficiency of transgenic IU human chorionic gonadotrophin (hCG;Farma-Lepori) and mated with males of theembryo banking have previously been re-

ported. same strain (2). Females were euthanized bycervical dislocation 48 h after hCG adminis-MATERIALS AND METHODStration and two-cell mouse embryos were ob-Animalstained by flushing the oviducts with M2

Transgenic mice used in this study weremedium (14). Embryos were collected in M2

produced as described in Ibanez et al. (10).medium and only morphologically normal

Briefly, a 14.5-kb caprine BLG transgene,two-cell embryos were selected for further

containing the entire transcription unit withstudy. Transgenic and F2 embryos were pro-

6.1 and 3.7 kb of the 5* and the 3* flankingcessed in parallel.

regions, respectively, was microinjected intopronuclear stage B6CBAF2 embryos. Four Freezing/Thawing Protocollines of transgenic mice were obtained, car-

Two-cell embryos were frozen in M2 me-rying 2–3 (Tg23), 2–4 (Tg58), and 1 (Tg101

dium containing 1.5 M propylene glycoland Tg158) copies of the transgene. The pres-

(PROH, Fluka) and 0.1 M sucrose (Merck) intoence of the exogenous protein was detected

0.25 ml plastic straws (2 1 133 mm; EMB,in the milk of transgenic females from lines

Spain) in horizontal position, in a computer-Tg23, Tg58, and Tg158, at levels of 0.2–0.5,

controlled freezer (Model Kryo-10; Planner0–0.02, and 0.3–0.4 mg/ml, respectively. No

Products Ltd., Sunbury-on-Thames, UK).cBLG expression was observed in animals

Cryoprotectant was added in two steps: 10from transgenic line Tg101.

min of incubation in a solution of 1.5 M PROHTransgenic lines were propagated by back-

in M2 and 5 min of incubation in a solution ofcrossing founder animals to B6CBAF1 mice

1.5 M PROH and 0.1 M sucrose in M2, bothand subsequent brother–sister crosses. Ani-

steps at room temperature. Embryos weremals homozygous for the transgene were ob-

slowly cooled and rapidly thawed, followingtained after mating hemizygous transgenic

the cooling/warming rates published by Las-mice and confirmed by backcrossing to non-

salle et al. (11). Briefly, embryos were cooledtransgenic animals. Transgenic animals used

to 077C at a rate of 027C/min and, after ain this study as embryo donors were from the

holding period of 5 min, seeding was automati-fifth (G5; line Tg23) and the sixth (G6; lines

cally induced by a cooper anvil attached to aTg58, Tg101, and Tg158) generations.

termoelectric module. Slow cooling was con-As a control, hybrid B6CBAF1 animals (F1

tinued to 0307C, at a rate of 00.37C/min, andmice), resulting from female C57Bl/6J1male

embryos were held for 2 min at this tempera-CBA/Ca matings, were also used as donors of

ture for equilibration. Finally, embryos wereF2 embryos.

rapidly cooled to 01507C at a rate of 0357C/All mice were housed in a temperature-con-

min and then transferred directly into liquidtrolled room, exposed to a 12-h light–12-h

nitrogen for storage. Embryos were thawed fordark cycle, and allowed free access to food

40 s at room temperature in air (Ç3247C/min)and water.

and 40 s in a water bath at 307C (Ç157C/min).Embryo Collection Dilution and removal of cryoprotectant

were initially performed in two steps: 15 minTransgenic females (4 to 12 weeks old)from lines Tg23, Tg58, Tg101, and Tg158 and of incubation in a solution of 0.75 M PROH


292 IBANEZ ET AL.

and 1 M sucrose in M2, at room temperature, also cultured in vitro for 72 h under the sameconditions.followed by 15 min of incubation in M2 me-

dium, at 377C. However, in embryos fromtransgenic line Tg101, a four-step cryoprotec- Viability Criteria and Statistical Analysistant dilution protocol was also applied, due to

In vitro viability of cultured unmanipulated,the poor results obtained with the former. Infrozen/thawed, and cryoprotectant control em-this protocol, embryos were exposed to M2bryos was evaluated every 24 h by checkingsolutions with decreasing PROH concentra-their developmental capacity and morphologytions (1, 0.5, and 0 M) and increasing sucroseunder an stereoscopic microscope. Frozen/concentrations (0.05, 0.1, and 0.2 M), for 5thawed or cryoprotectant control embryosmin each at room temperature. The fourth stepshowing at least one morphologically normalconsisted of 5 min of incubation in M2 me-blastomere after treatment were considered vi-dium at 377C.able, as it has been previously reported that

Cryoprotectant Dilution Control ‘‘half-embryos’’ are able to develop in vitroand in vivo, yielding normal live pups (8, 17).To investigate potential effects of cryopro-

Recovery rate of frozen/thawed and cryo-tectant toxicity and osmotic changes duringprotectant control embryos was calculated ascryopreservation, embryos were exposed tothe percentage of thawed embryos that sur-freezing and thawing solutions for the samevived cryopreservation immediately aftertime as cryopreserved ones, but at room tem-thawing (0 h). Survival rate was calculated asperature, thus mimicking freezing/thawingthe percentage of cultured embryos (survivingtreatment without the effect of low tempera-embryos at 0 h) that developed into blastocyststures (cryoprotectant control embryos).after 72 h of in vitro culture.Cryoprotectant dilution protocol was in

Data were analyzed by Fisher’s exact test,concordance with that used in freezing/thaw-considering P õ 0.05 as statistically signifi-ing treatment: a two-step protocol was usedcant.for transgenic embryos from lines Tg23 and

Tg58, while a four-step protocol was used forRESULTStransgenic embryos from line Tg101. Cryo-

protectant dilution control treatment could not Embryo Productionbe applied to Tg158 group due to the lownumber of animals, and thus embryos, avail- Production of two-cell embryos after super-

ovulation treatment and mating of transgenicable from this transgenic line.Data from cryoprotectant control F2 embryos female mice was studied and compared to em-

bryo production of F1 females (Table 1). Inwere obtained from studies previously per-formed by our group, where dilution of cryo- all transgenic lines, the number of two-cell

embryos produced per female was signifi-protectant was performed in two steps (18).cantly lower than in F1 females. Statistically

Embryo Culture significant differences in embryo productionwere also observed between the fourAs a control of in vitro developmental ca-

pacity, unmanipulated two-cell embryos from transgenic lines: females from line Tg101 pro-duced the highest number of embryos (11.7the four lines of transgenic mice and F2 em-

bryos were cultured for 72 h in M16 medium embryos per female) while females from lineTg158 produced the lowest (1.4 embryos per(25) under paraffin oil in standard conditions

(377C, 5% CO2 in air). Transgenic and F2 female). No differences were found betweenlines Tg23 and Tg58 (5.3 and 4.7 embryosembryos surviving the freezing/thawing or the

cryoprotectant dilution control treatment were per female, respectively).



TABLE 1Two-Cell Embryo Production after Standard Superovulation Protocol

and Mating of Transgenic and F1 Female Mice

F1 Tg23 Tg58 Tg101 Tg158

Number of experiments 5 5 8 4 4Number of females 32 51 85 43 33Number of two-cell embryos obtained 580 270 402 505 47Number of embryos/female 18.1a 5.3b 4.7b 11.7c 1.4d

a–d Values with different superscripts differ between columns (P õ 0.0001).

Embryo Developmental Capacity in Vitro Tg101, and Tg158, respectively, and 71 two-cell F2 embryos were frozen in several experi-As a control of embryo development inments carried out on different days.vitro, two-cell transgenic embryos (Tg23,

The number of cryopreserved embryos thatTg58, Tg101, and Tg158) and F2 embryoswere thawed following the two-step cryopro-were cultured for 72 h and their viability wastectant dilution protocol were 59, 59, 23, andcompared (Table 2). No statistically signifi-12 for transgenic lines Tg23, Tg58, Tg101,cant differences were observed in the in vitroand Tg158, respectively, and 71 for F2 group.development to blastocyst stage between F2Recovery rates and survival rates after 72 h(84.1%), Tg23 (82.3%), Tg58 (75.4%), andof in vitro culture are shown in Table 3. ATg101 (71.9%) embryos. However, a greatervery low recovery rate (39.1%) was observedvariability between experiments was found infor Tg101 embryos compared with all otherthe proportion of embryos reaching the blasto-groups and survival rate after 72 h of in vitrocyst stage in embryos from transgenic linesculture (11.1%) was also significantly lowerTg23 and Tg101 than in embryos from F1 andthan for all other groups, except for Tg158Tg58 females (Table 2).embryos (18.2%).Due to the low number of embryos avail-

Due to this low recovery rate of Tg101 em-able from transgenic line Tg158, only one rep-bryos after thawing using the two-step cryo-licate of in vitro culture was performed andprotectant dilution protocol, an alternativethus this group was not included in the statisti-four-step dilution protocol was applied to thiscal analysis.group. A significant improvement was found

Embryo Freezing and Thawing both in the recovery rate (91.1 vs 39.1%;P õ 0.0001) and in the survival rate up toA total of 152, 160, 267, and 29 two-cell

transgenic embryos from lines Tg23, Tg58, blastocyst stage (58.5 vs 11.1%; P õ 0.05)

TABLE 2Developmental Capacity of Unmanipulated Embryos after 72 h of in Vitro Culture

F2 Tg23 Tg58 Tg101 Tg158

Number of experiments 5 4 5 4 1Number of embryos 69 85 61 64 18Survival rate % 84.1a 82.3a 75.4a 71.9 a 44.4b

(SD value) (11.4) (27.2) (21.1) (48.1) (0)

a Not significantly different.b Not included in the statistical analysis.


294 IBANEZ ET AL.

TABLE 3Recovery and Survival Rates of Frozen / Thawed Embryos

F2 Tg23 Tg58 Tg101(2)1 Tg101(4)1 Tg158

Number of experiments 5 3 4 2 4 2Number of embryos 71 59 59 23 56 12Recovery rate % 83.1a,b 76.3a 81.4a,b 39.1c 91.1b 91.7a,b

(SD value) (5.1) (10.7) (13.4) (19) (8.8) (11.7)Survival rate % 72.9 a 64.4a 77.1a 11.1b 58.8a 18.2b

(SD value) (13.4) (36.5) (10) (14.1) (38) (23.5)

a–c Values with different superscripts differ between columns (P õ 0.05).1 Tg101 (2) and Tg101 (4) indicate the two different protocols for cryoprotectant dilution used in cryopreservation

of this embryo group (two- or four-step protocol, respectively).

(Table 3). The recovery rate for Tg101 em- posed to freezing and thawing solutions forthe same time as cryopreserved embryos,bryos that underwent the four-step cryoprotec-

tant dilution protocol was now equivalent to though at room temperature. The two-steprecovery rates for F2, Tg58, and Tg158 em- cryoprotectant dilution protocol was used withbryos and even higher than for Tg23 embryos F2, Tg23, and Tg58 embryos, while in group(Table 3). After 72 h of in vitro culture, sur- Tg101 the four-step protocol was applied.vival rates were similar between all groups, No differences were detected in the recov-except for Tg158 embryos, which showed the ery rate of transgenic and F2 embryos afterworst developmental capacity. exposure to freezing and thawing solutions

Again, a high degree of variability between (Table 4). However, a significantly lower per-experiments was observed in the survival rates centage of Tg23 embryos than of F2 and Tg58of embryos from group Tg23 and from group embryos (68.3 vs 83.5 and 88%, respectively)Tg101, both when using the two-step and the developed into blastocysts after 72 h of infour-step cryoprotectant dilution protocol. In vitro culture. As in all other treatments, sur-this case, a high degree of variability was also vival rates of Tg23 and Tg101 embryosobserved in survival rates of Tg158 embryos showed a high degree of variability between(Table 3). different experiments (Table 4).

Comparisons of the survival rates after 72 Recovery rates of frozen/thawed (Table 3)h of in vitro culture of unmanipulated (Table and cryoprotectant control embryos (Table 4)2) and frozen/thawed embryos (Table 3) were were compared for each group of embryosperformed for all individual groups, except for (except for Tg158 embryos, to which no cryo-Tg158 embryos (insufficient data for statisti- protectant dilution control treatment was ap-cal analysis). In vitro development up to blas- plied) to determine embryo capacity to re-tocyst stage was similar in unmanipulated and cover from exposure to low temperatures. Infrozen/thawed embryos from all groups, ex- all groups, cryoprotectant control embryoscept for group Tg23 (P õ 0.05) and group showed a higher recovery rate than frozen/Tg101 (P õ 0.001) when thawed using the thawed embryos (P õ 0.001), except fortwo-step dilution protocol, in which freezing/ Tg101 embryos that underwent the four-stepthawing process significantly reduced their de- cryoprotectant dilution protocol after thawing.velopmental capacity. Survival rates up to blastocyst stage ofCryoprotectant Control Embryos cryoprotectant control embryos (Table 4) were

statistically compared to those of unmanipu-Transgenic embryos from groups Tg23,Tg58, and Tg101 and F2 embryos were ex- lated (Table 2) and of frozen/thawed embryos



TABLE 4Recovery and Survival Rates of Cryoprotectant Control Embryos

F2 Tg23 Tg58 Tg101(4)1

Number of experiments 3 3 3 3Number of embryos 162 60 50 50Recovery rate % 97.5a 100 a 100 a 100 a

(SD value) (1.9) (0) (0) (0)Survival rate % 83.5b 68.3c 88b 76b,c

(SD value) (15.4) (46.5) (19) (53.4)

a Not significantly different.b,c Values with different superscripts differ between columns (P õ 0.05).1 Number in parentheses indicates the four-step cryoprotectant dilution protocol used with this embryo group.

effect of the site of transgene integration can-(Table 3), respectively, and no significant dif-not be discounted.ferences were detected in any of the groups.

In spite of the differences observed in em-DISCUSSION bryo production after superovulatory treat-

A significant decrease in two-cell embryo ment, two-cell embryos obtained from F1 fe-production after standard superovulation treat- males and from Tg23, Tg58, and Tg101 fe-ment of females was observed in all lines of males showed similar developmental capacitycBLG transgenic mice with respect to control up to the blastocyst stage when cultured inF1 females, and differences were also found vitro for 72 h. Embryos from line Tg158 werebetween transgenic lines. Although the high- not included in the statistical analysis as onlyest two-cell embryo production was obtained one replicate could be performed contempora-from Tg101 females, which do not express the neously. However, our impression was thattransgene in their milk, an effect of cBLG this group showed the worst developmentalexpression on exogenous hormonal induction capacity when cultured in vitro.of superovulation would be difficult to ex- Several factors have been reported to causeplain. Moreover, no correlation exists between embryo damage during freezing/thawing pro-the level of cBLG expression and response to cess, and among them cytotoxicity of cryopro-superovulation in these four transgenic lines. tectant, osmotic shock, severe dehydration,

Differences in the reproductive perfor- pH changes in the freezing solutions and intra-mance of females from various mouse strains cellular ice formation seem to be the mostand stocks after gonadotrophin treatment have important (for review see 1, 5, and 13). Whenbeen reported in several studies and the geno- cryopreservation conditions are adjusted totype of the donor female has been identified each particular cell type, detrimental effectsas the major factor contributing to this varia- of such factors can be considerably reducedtion (19, 20, 22, 23). As transgenic lines are and limited to the freezing/thawing process.expanded by brother–sister crosses, inbreed- No long-term consequences seem to exist, asing for several generations (five/six in the embryos that survive cryopreservation arepresent study) results in genetic differences able to develop in in vitro culture as unfrozenamong the transgenic lines. Therefore, the ones. As we routinely freeze F2 two-cellvariability in two-cell embryo production ob- mouse embryos with satisfactory results, theserved among transgenic and F1 females same cryopreservation protocol (9, 12, 18)could probably be attributed to the differences was initially applied for freezing two-cell

transgenic embryos. Moreover, embryo cryo-in the genetic background. Nevertheless, an


296 IBANEZ ET AL.

preservation at this early stage allowed subse- immediately after embryo exposure to the firstsolution for the removal of cryoprotectant, andquent in vitro viability studies.this is consistent with an osmotic shockRecovery rates and survival rates up to blas-caused by a very slow exit of intracellulartocyst stage, equivalent to those obtained forcryoprotectant and a rapid flow of water intoF2 embryos, were achieved for transgenic em-the cytoplasm. Second, and most important,bryos from lines Tg23 and Tg58 after thawing.when a four-step cryoprotectant dilution pro-Moreover, survival rates up to blastocyst stagetocol was applied to this group, with a moreof frozen/thawed F2, Tg23, and Tg58 embryosgradual removal of cryoprotectant, a signifi-were equivalent to those of their unfrozencant increase in both the recovery rate and thecounterparts (though differences were de-survival rate of frozen/thawed Tg101 embryostected in Tg23 group, the high variability be-was observed. Moreover, the in vitro develop-tween experiments could account for this sta-mental capacity of Tg101 embryos that under-tistically discordant results).went this alternative protocol was equivalentAlthough the survival rate of frozen/thawedto that of their unfrozen counterparts.Tg158 embryos after 72 h of in vitro culture

Exposure of transgenic and F2 embryos towas significantly lower than that of frozen/freezing and thawing solutions without cool-thawed F2, Tg23, and Tg58 embryos, recov-ing did not have any detrimental effect onery rates were similar. Thus, their apparent

low developmental capacity when cultured in embryo viability, when the appropriate cryo-protectant dilution protocol was applied, asvitro seems to be the cause for this decrease,

rather than an effect of the freezing/thawing has been reported in previous studies (6, 18).Recovery rates and survival rates of cryopro-treatment.tectant control transgenic and F2 embryosA much more detrimental effect of cryopres-were similar and equivalent to survival rateservation, however, was observed for transgenicof their unfrozen counterparts. Again, differ-embryos from line Tg101. These transgenicences detected in the survival rate of cryopro-embryos showed not only a lower recovery ratetectant control Tg23 embryos, when comparedafter thawing, but also, and most surprising, ato F2 and Tg58 embryos, are most probablylower survival rate after 72 h of in vitro culture.due to the high interexperiment variability,Moreover, when comparing the developmentalrather than to a detrimental effect of this treat-capacity of unfrozen and frozen/thawed Tg101ment, as survival rates of cryoprotectant con-embryos, a significant decrease was observedtrol Tg23 embryos were equivalent to thosein these later group. Therefore, the reducedof unmanipulated ones.postthaw development of Tg101 embryos may

reflect a greater sensitivity of these transgenic The embryo recovery rate was lower in fro-zen/thawed embryos than in cryoprotectantembryos to cryoinjury.control ones, thus indicating an effect of cool-Cellular properties of embryos, such as theing and warming on embryo viability. Factorssize of blastomeres, the permeability of cellsuch as the mechanical damage caused by themembranes to cryoprotectants, and the em-ice front and/or partial dehydration as a resultbryo sensitivity to cold shock, intracellularof extracellular water freezing, which in-freezing, or cryoprotectant toxicity, may in-creases osmolality outside of the cell, couldfluence their ability to survive cryopreserva-account for this effect (1, 24). However, thistion (7, 16). The low recovery and survivaldoes not compromise developmental capacityrates of frozen/thawed Tg101 embryos mightin surviving embryos, as no differences werebe attributed to a poor membrane permeabilityfound between survival rates of cryoprotectantto cryoprotectant and, though no experimentalcontrol and frozen/thawed embryos.data are available, two observations support

this hypothesis. First, embryo lysis occurred In summary, data from this study indicate



ods in Mammalian Embryology’’ (J. C. Daniel,that differences exist in the response of cBLGEd.), pp. 86–116. Freeman, San Francisco, CA,transgenic females to the standard superovula-1971.tory treatment and also in the response to cryo-

3. Cheng, S. S., and Costantini, F. Morula decompactionpreservation of transgenic embryos produced (mdn), a preimplantation recessive lethal defect inby these females. However, slight modifica- a transgenic mouse line. Dev. Biol. 156, 265–277tions in the freezing/thawing protocol allowed (1993).

4. Covarrubias, L., Nishida, Y., and Mintz, B. Earlyus to achieve successful cryopreservation ofpostimplantation embryo lethality due to DNA re-two-cell embryos from all transgenic lines.arrangements in a transgenic mouse strain. Proc.Variability in the ovulation rate after gonad-Natl. Acad. Sci. USA 83, 6020–6024 (1986).

otrophin treatment of females and in the post- 5. Critser, J. K., Arneson, B. W., Aaker, D. V., Huse-thaw development of cryopreserved embryos Benda, A. R., and Ball, G. D. Factors affectinghas been previously reported for various strains the cryosurvival of mouse two-cell embryos. J.

Reprod. Fertil. 82, 27–33 (1988).and stocks of mice, and it is mainly attributed6. Damien, M., Luciano, A. A., and Peluso, J. J. Pro-to genotype (7, 19–23). Therefore, differences

panediol-induced alterations in membrane integ-in the genetic background between the fourrity, metabolism and developmental potential of

transgenic lines used in our study could account mouse zygotes. Hum. Reprod. 4, 969–974 (1989).for the variations observed, both in the two- 7. Dinnyes, A., Wallace, G. A., and Rall, W. F. Effectcell embryo production by transgenic females of genotype on the efficiency of mouse embryo

cryopreservation by vitrification or slow freezingand in the ability of transgenic embryos to sur-methods. Mol. Reprod. Dev. 40, 429–435 (1995).vive cryopreservation. However, we cannot ex-

8. Gimenez, C., Egozcue, J., and Vidal, F. Cytogeneticclude a potential effect of the site of transgenesexing of mouse embryos. Hum. Reprod. 8, 470–

integration, which is also different in the four 474 (1993).transgenic lines. 9. Grossmann, M., Egozcue, J., and Santalo, J. Cryo-

preservation of zona-free mouse embryos. Cryo-The results obtained in this study may haveLett. 15, 103–112 (1994).important implications for the maintenance of

10. Ibanez, E., Folch, J. M., Vidal, F., Coll, A., Santalo,transgenic lines as frozen embryo stocks. CareJ., Egozcue, J., and Sanchez, A. Expression ofshould be taken to assess cryopreservationcaprine b-lactoglobulin in the milk of transgenic

conditions on each transgenic line and to es- mice. Transgenic Res. 6, 69–74 (1997).tablish the appropriate protocol, before the 11. Lassalle, B., Testart, J., and Renard, J. P. Human em-

bryos features that influence the success of cryo-generation of a transgenic embryo cryobank.preservation with the use of 1,2-propanediol. Fer-This will ensure successful recovery of cryo-til. Steril. 44, 645–651 (1985).preserved valuable transgenic animals.

12. MartıB , M., Grossmann, M., Santalo, J., Egozcue, J.,and Ponsa, M. Characteristics of actin fibers andACKNOWLEDGMENTSultrastructure of the contact regions involved in

This research was financially supported by CICYT the separation of blastomeres of two-cell mouse(Spain) (AGF 96/1093CO2/02) and Institut de Cultura embryos, frozen-thawed without the zona pellu-Joan-Gil Albert (Diputacio d’Alacant, Spain). Authors cida. Cryobiology 34, 94–106 (1997).thank Dr. A. Sanchez, Dr. J. M. Folch and Dr. A. Coll 13. Pollard, J. W., and Leibo, S. P. Chilling sensitivityfor their collaboration in the generation of the transgenic of mammalian embryos. Theriogenology 41, 101–lines used in this study. 106 (1994).

14. Quinn, P., Barros, C., and Whittingham, D. G. Preser-REFERENCES vation of hamster oocytes to assay the fertilizing

capacity of human spermatozoa. J. Reprod. Fertil.1. Ashwood-Smith, M. J., Morris, G. W., Fowler, R.,66, 161–168 (1982).Appleton, T. C., and Ashorn, R. Physical factors

15. Radice, G., Lee, J. L., and Costantini, F. Hb58, anare involved in the destruction of embryos andinsertional mutation affecting early postimplanta-oocytes during freezing and thawing procedures.tion development of the mouse embryo. Develop-Hum. Reprod. 3, 795–802 (1988).ment 111, 801–811 (1991).2. Biggers, J. D., Whitten, W. K., and Whittingham,

D. C. The culture of mouse ova in vitro. In ‘‘Meth- 16. Rall, W. F. Cryopreservation of oocytes and em-


298 IBANEZ ET AL.

bryos: Methods and applications. Anim. Reprod. freezing variables. Biol. Reprod. 37, 1121–1128(1987).Sci. 28, 237–245 (1992).

22. Spearow, J. L. Major genes control hormone-induced17. Rulicke, Th., and Autenried, P. Potential of two-cellovulation rate in mice. J. Reprod. Fertil. 82, 787–mouse embryos to develop to term despite partial797 (1988).damage after cryopreservation. Lab. Anim. 29,

23. Spearow, J. L., and Bradford, G. E. Genetic variation320–326 (1995).in spontaneous ovulation rate and LH receptor in-18. Sandalinas, M., Grossmann, M., Egozcue, J., andduction in mice. J. Reprod. Fertil. 69, 529–537Santalo, J. Blastomere disaggregation of cryopre-(1983).served two-cell zona-free mouse embryos. Cryo-

24. Van Oss, C. J., Giese, R. F., and Norris, J. InteractionLett. 15, 343–352 (1994).between advancing ice fronts and erythrocytes.

19. Schmidt, P., Hansen, C. T., and Wildt, D. E. ViabilityCell Biophys. 19, 253–261 (1992).

of frozen–thawed mouse embryos is affected by 25. Whittingham, D. G. Culture of mouse ova. J. Reprod.genotype. Biol. Reprod. 32, 507–514 (1985). Fertil. (Suppl. 1) 14, 7–21 (1971).

20. Schmidt, P., Monfort, S. L., and Wildt, D. E. Preg- 26. Whittingham, D. G., Leibo, S. P., and Mazur, P. Sur-nant mare’s serum gonadotropin source influences vival of mouse embryos frozen to 01967C andfertilization and fresh or thawed embryo develop- 02697C. Science 178, 411–417 (1972).ment, but the effect is genotype specific. Gamete 27. Wilmut, I. The effect of cooling rate, warming rate,Res. 23, 11–20 (1989). cryoprotective agent and stage of development on

21. Schmidt, P., Schiewe, M. C., and Wildt, D. E. The survival of mouse embryos during freezing andthawing. Life Sci. 11, 1071–1079 (1972).genotypic response of mouse embryos to multiple


Cryopreservation of Caprine β-Lactoglobulin Transgenic Mouse Embryos

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