Production of transgenic mice from cryopreserved fertilized ova.

Cryopreserved fertilized mouse ova were used to generate transgenic mice via micromanipulation. Five-DNA constructions were injected into a total of 1,052 cryopreserved ova, of which 683 (65%) survived the injection and were transferred into recipients. Of 35 recipients, 66% became pregnant and littered a total of 88 pups. As controls, these DNA constructions were also injected into 1,123 fresh ova, of which 744 (66%) survived and were transferred. Of 42 recipients, 79% became pregnant and littered a total of 167 pups. That is, 22% of fresh ova that were transferred developed into live pups, whereas only 13% of cryopreserved ova did so. Of the pups born, 42 of the 167 (25%) produced from fresh ova were transgenic, and 28 of the 88 (32%) produced from cryopreserved ova were transgenic. In terms of the injected ova that had been transferred, 5.6% of the 744 fresh and 4.1% of the 683 frozen ova developed into transgenic mice. These data indicate that the efficiency of production of transgenic mice from cryopreserved ova is close to that from fresh ova. That observation and the fact that cryopreserved ova allow more efficient utilization of animals suggest that cryopreserved ova can be used instead of fresh ova to produce transgenic mice.     

Cryogenic effect of antifreeze protein on transgenic mouse ovaries and the production of live offspring by orthotopic transplantation of cryopreserved mouse ovaries

The purpose of the present study was to evaluate the cryogenic effect of antifreeze protein (AFP) on transgenic mouse ovaries which is expressed AFP type III from Ocean pout and the production of live offspring by orthotopic transplantation of cryopreserved mouse ovaries. In this study, whole transgenic and nontransgenic mouse ovaries were vitrified with 20% DMSO and 20% EG in M2 medium supplemented with 0.5 M sucrose. All vitrified and toxicity control and fresh ovaries were transplanted orthotopically into ovariectomized recipients bilaterally. For fresh ovaries transplantation, 5 mice delivered litters of 18 and 19 live pups in first and second matings, respectively. For toxicity control of chemicals, 6 mice delivered litters of 22 and 23 live pups. For nontransgenic mouse ovaries (vitrified) transplantation, 7 mice delivered litters of 22 and 23 live pups. For transgenic mouse ovaries (vitrified) transplantation, 10 mice delivered litters of 35 and 37 live pups. Litter sizes from pups of freshly transplanted ovaries were not significantly different from AFP-transplanted transgenic ovaries but those from nontransgenic-transplanted ovaries were significantly different from the AFP-transplanted transgenic ovaries group (P < 0.05). In this study, for the first time, it was shown that the ovarian tissue of AFP transgenic mice was protected from cryopreservation by vitrification. These results demonstrate that a normal reproductive lifespan can be restored by orthotopic transplantation of AFP transgenic-vitrified ovary.     

In vivo development of vitrified rat embryos: effects of timing and sites of transfer to recipient females

In cryopreserved rat embryos, survival rates obtained in vitro are not always consistent with the rates obtained in vivo. To determine the optimal conditions for in vivo development to term, rat embryos at the 4-cell, 8-cell, and morula stages were vitrified in EFS40 by a one-step method and transferred into oviducts or uterine horns of recipients at various times during pseudopregnancy. Vitrified and fresh 4-cell embryos only developed after transfer into oviducts of asynchronous recipients on Days -1 to -2 of synchrony (i.e., at a point in pseudopregnancy 1-2 days earlier than the embryos). Approximately half the vitrified embryos transferred into oviducts on Day -1 developed to term, but only a minority of embryos, whether vitrified (10%-34%) or fresh (24%-33%), transferred at later times did so, suggesting that this may not be the most suitable stage for cryopreservation. Very few 8-cell embryos, either vitrified or fresh, developed when transferred into oviducts on Day 0 to -0.5. However, when transferred into uterine horns, high proportions of vitrified 8-cell embryos ( approximately 63%) developed to term in reasonably synchronous recipients (Day 0 to -0.5) but not in more asynchronous ones (6%; Day -1). A majority of vitrified morulae also developed to term (52%-68%) in a wider range of recipients (Days 0 to -1), the greatest success occurring in recipients on Day -0.5. Similar proportions of vitrified and fresh 4-cell embryos, 8-cell embryos, and morulae developed to term when appropriate synchronization existed between embryo and recipient. Thus, vitrification of preimplantation-stage rat embryos does not appear to impair their developmental potential in vivo.     

A simple and rapid method for cryopreservation of mouse 2-cell embryos by vitrification: Beneficial effect of sucrose and raffinose on their cryosurvival rate

Survival of mouse 2-cell embryos was evaluated after exposure to 1.38, 2.75 or 5.5 M single cryoprotectants [dimethylsulphoxide (DMSO), acetamide (Ac) and propylene glycol (PG)], components frequently utilized as a vitrification solution, for 0.5, 1, 2 and 10 minutes at room temperature prior to vitrification. More than 80 % of the treated embryos developed to normal blastocysts in culture, after exposure to 1.38-2.75 M of each reagent for 0.5 minutes, although Ac tended to provide with have a deleterious effect on their survival. When the embryos were vitrified with solutions containing DP (2.75 M DMSO and 2.75 M PG) plus 0, 0.5 and 1.0 M Ac after a 0.5-minute exposure, their in vitro survival rates to the blastocysts were 44, 41 and 37%, respectively, showing no significant difference among them (x(2)=0.1-0.6, P>0.05). This indicates that the presence of Ac is not always needed for vitrifying mouse 2-cell embryos. Embryos, that had been vitrified with DP solution supplemented with 1.0 M sucrose (DPS) after a 0.5- minute exposure, exhibited significantly higher in vitro survival rate (82%) than those vitrified with DP (44%) (x(2)=23.4, P<0.001). Similar high survival rate (81%) was obtained when they were vitrified with DP plus 0.16 M raffinose (DPR) (x(2)=28.3, P<0.001). In vivo survival of embryos vitrified with DPS or DPR after a 0.5-minute exposure was both 49%, and there was no significant difference comparing to the unvitrified control group (60%). This method is rapid, efficient and reliable, and thus may be of practical use for cryopreserving mouse 2-cell embryos.     

昆明小鼠精子冷冻的研究（简报）

胚胎工程技术是动物品种、品系培育,种质资源保存及转基因动物制备、保种的重要手段。配子的冷冻保存技术目前广泛应用于胚胎工程。和胚胎冷冻相比小鼠精子冷冻技术方便、高效尤其适用于转基因及突变系小鼠的保种。成功的精子冷冻要求复苏后通过体外受精(IVF)获得胚胎,再移植入受     

Vitrification and transfer of cynomolgus monkey (Macaca fascicularis) embryos fertilized by intracytoplasmic sperm injection

Protocols for cryopreservation of monkey embryos are not well established. The objective of the current study was to determine the efficacy of the polypropylene strip method for cryopreserving cynomolgus monkey embryos. Cynomolgus monkey embryos, 63 and 56 at the 4- to 8-cell and 56 blastocyst stages, respectively, were produced by intracytoplasmic sperm injection and in vitro culture, and vitrified using a polypropylene strip. For these two stages, 95 and 86% survived after thawing and pregnancy rates were 29.2% (7 pregnant females/24 recipients, with three live births) and 0% (n = 16 recipients). These were apparently the first live births obtained from embryos fertilized by ICSI. In conclusion, 4- to 8-cell preimplantation cynomolgus monkey embryos were successfully cryopreserved using a polypropylene strip.     

Production of transgenic mice from vitrified pronuclear-stage embryos.

Cryopreservation of pronuclear-stage embryos would be useful for transgenic technology and genome preservation purposes. We compared a novel vitrification technique (solid surface vitrification, SSV) with another vitrification method in straws for cryosurvival and to generate transgenic progeny from cryopreserved mouse zygotes following microinjection. The SSV solution consisted of 35% ethylene glycol (EG), 5% polyvinyl-pyrrolidone (PVP), and 0.4 M trehalose in M2 supplemented with 4 mg/ml BSA; the in straw vitrification solution was 7 M EG in M2 plus BSA. In experiment I, we compared the effect of the vitrification solutions alone, without cooling. No reduction was detected in survival and cleavage rates. In experiment II, SSV yielded a significantly higher percentage of morphologically normal zygotes (96%) that also cleaved at significantly higher rates (80%) when compared to that following "in straw" vitrification (68 and 66%, respectively). Cleavage rate in the non-vitrified control group (93%) was significantly higher than that of both vitrified groups. Following embryo transfer, there was no difference in the rate of pups obtained from the SSV, "in straw" vitrified, and control groups (97/457, 21%; 15/75, 20% and 56/209, 27%, respectively). In experiment III, SSV vitrified and fresh embryos were used for pronuclear DNA injection. Survival rate of vitrified embryos after microinjection was reduced compared to nonvitrified ones (64 vs. 72%, respectively; P < 0.05); however, development to two-cell stage was not different (76 vs. 72%, respectively). Following embryo transfer of vitrified vs. fresh microinjected embryos, in both cases 10% live pups were generated, including transgenic pups. The results demonstrated that the efficiency of generating transgenic pups from SSV vitrified pronuclear zygotes is comparable to that from fresh embryos.     

First pregnancy and live birth from vitrified rabbit oocytes after intraoviductal transfer and in vivo fertilization

Intraoviductal oocyte transfer in combination with in vivo fertilization has arisen as an alternative method to induce pregnancies from cryopreserved oocytes in rabbits. In this study, offspring were obtained for the first time from vitrified rabbit oocytes using this technique. In all the experiments, recipients were artificially inseminated 9 hours before oocyte transfer. Cryopreserved (vitrified and slow-frozen) and noncryopreserved (fresh) oocytes were transferred into both oviducts, which were immediately closed using cyanoacrylate tissue adhesive to block the entry of the recipient's own oocytes. Three transferred group females that received vitrified oocytes became pregnant and delivered a total of nine live young naturally. The results revealed that there were no differences in the live birth rate between vitrified and slow-frozen oocytes (5.5% and 4.4%, respectively). When fresh oocytes were transferred, this rate increased to 19.2%, whereas in the control females (nontransferred) the rate of offspring obtained was 71.4%. This is the first reported result of the development to term of vitrified rabbit oocytes and suggests that an in vivo environment could help improve the results of oocyte cryopreservation.     

胞浆内精子注射技术生产小鼠

以piezo操作系统为技术支撑 ,在掌握小鼠卵母细胞胞浆内精子注射技术 (ICSI)的基础上 ,进行了ICSI技术生产试管小鼠的尝试。来自成年昆明 (KM)小鼠附睾尾的新鲜精子 ,剪切去尾后 ,直接将精子头注射到B6D2F1小鼠卵母细胞质中 ,注射后 1h ,83.3%的卵母细胞存活。6h时 ,84.0 %的成活卵子成功受精 ,形成原核 ,排出PB2 体外培养的ICSI胚胎 ,卵裂率 (98%vs 94.7% )和 4-细胞期胚胎比率 (89.5%vs 92.1% )均与培养的体内受精卵没有差异 (P >0.05 ) ;但是 ,桑椹胚(63.8%vs84.2% )和囊胚发育率 (25.7%vs68.4% )极显著地 (P <0.01)低于对照组。120枚原核期胚胎移植给 7只假孕受体后 ,4只受孕小鼠共产出 28只ICSI小鼠 (23.3% )。健康成年的 25只ICSI小鼠都没有明显的生理和行为异常。随机选择其中的 20只小鼠 ,分别进行ICSI小鼠间、ICSI与KM小鼠间共 12组的交配 ,结果所有雌鼠妊娠产仔。在成功建立小鼠ICSI技术的基础上,成功获得了我国的首例ICSI小鼠,并且证明这些ICSI小鼠都具有正常的繁殖后代的能力。     

Production of transgenic miniature pigs by pronuclear microinjection

Miniature pig is an attractive animal for a wide range of research fields, such as medicine and pharmacology, because of its small size, the possibility of breeding it under minimum environmental controls and the physiology that is potentially similar to that of human. Although transgenic technology is useful for the analysis of gene function and for the development of model animals for various diseases, there have not yet been any reports on producing transgenic miniature pig. This study is the first successful report concerning the production of transgenic miniature pig by pronuclear microinjection. The huntingtin gene cloned from miniature pig, which is a homologue of candidate gene for Huntington's disease, connected with rat neuron-specific enolase promoter region, was injected into a pronucleus of fertilized eggs with micromanipulator. The eggs were transferred into the oviduct of recipient miniature pigs, whose estrus cycles were previously synchronized with a progesterone analogue. A total of 402 injected eggs from 171 donors were transferred to 23 synchronized recipients. Sixteen of them maintained pregnancy and delivered 65 young, and one resulted in abortion. Five of the 68 offspring (three of which were aborted) were determined to have transgene by PCR and Southern analysis. The overall rate of transgenic production was 1.24% (transgenic/injected eggs). This study provides the first success and useful information regarding production of transgenic miniature pig for biomedical research.     

Intracytoplasmic Sperm Injection Using Cryopreserved, Fixed, and Freeze-dried Sperm in Eggs of Nile Tilapia

Gamete preservation techniques are essential in animal husbandry as well as in assisted reproduction for humans. In this research we attempted to use 3 different sperm preservation techniques in combination with newly developed techniques for intracytoplasmic sperm injection (ICSI) to fertilize eggs of a teleost fish, the Nile tilapia (Oreochromis niloticus). Of 47 eggs injected with fresh sperm, 11 (23%) were fertilized, 5 developed abnormally, and 4 developed normally and hatched; from these, one grew to adulthood. Nuclear DNA content of 4 of the abnormal embryos indicated that they were diploid. Flow cytometric analysis of a blood sample from the surviving ICSI fish collected 2 months after fertilization indicated that the fish was diploid. Of 45 eggs injected with cryopreserved sperm, 9 (20%) developed to the blastula stage. Of 40 eggs injected with sperm preserved in 70% methanol, none were fertilized. No injections were possible with freeze-dried Nile tilapia sperm owing to technical difficulties during manipulation. Although the findings described here are limited, they provide the first steps toward using sperm preservation methods in addition to cryopreservation for fertilization in fishes.     

Banking of embryos of mutated, paralytic tremor rabbit by means of vitrification

Cryopreservation enables banking of embryos for future use in medicine and in animal breeding. It also enables protection of germ plasm of endangered species and unique strains or lanes of laboratory animals. This paper describes an example of employing a vitrification method for banking of embryos of a unique lane of rabbit. The paralytic tremor (pt) rabbit is an X-linked recessive mutant lane of the Chinchilla breed characterized by hypomyelination of the central nervous system. In order to obtain a sufficient number of embryos, pt females were subjected to superovulation and surgical embryo collection. All suitable embryos were vitrified in 0.25 mL insemination straws in a modified EFS vitrification solution comprised of ethylene glycol (40%), Ficoll 70 (18%) and sucrose (0.3 M) in Hepes buffered TCM medium containing 20% fetal calf serum. In order to assess the efficiency of the vitrification procedure, a representative portion of vitrified embryos was warmed after a period of storage. Warmed embryos were subjected to in vitro culture for 72 h or were transferred to the uterus of synchronized recipients. The majority of the 141 warmed embryos survived vitrification and 100/141 (71%) developed to the blastocyst stage. Moreover, out of an additional 34 warmed embryos transferred to four recipients, eight (23.5%) developed to term and seven live pups were born. Six of the rabbit pups exhibited paralytic tremor symptoms typical for the pt lane. Although the overall efficiency of the vitrification method was lower compared with the effects usually achieved for 'healthy' embryos, results presented confirm the real possibility of the future restoration of the colony of pt rabbit, if sufficient number of embryos are cryopreserved.     

转hDAF基因小鼠的构建(英文)

本工作构建了含有ｈＤＡＦ基因的转基因小鼠,以便研究ｈＤＡＦ基因能否消除异种器官移植中的排斥反应。 采用ＤＮＡ重组的方法构建ｈＤＡＦ基因的表达载体ｐＳＰ６４ＨＰ（Ｆｉｇ．１）。通过受精卵显微注射,将其中的目的基因片段,转移到小鼠体内,建立转基因小鼠。再通过Ｄｏｔ ｂｌｏｔｔｉｎｇ和Ｓｏｕｔｈｅｒｎ ｂｌｏｔｔｉｎｇ杂交方法对出生小鼠的基因组特征进行查证。 连续两次对直接裂解菌液做ＰＣＲ扩增,筛选出重组质粒（Ｆｉｇ．２＆３）,酶切图谱（Ｆｉｇ．４）和Ｓｏｕｔｈｅｒｎ杂交（Ｆｉｇ．５）分析结果与预期吻合,出现预期条带;小鼠受精卵注射后存活比率为７７．９％,受精卵的发育率为３．４％,出生小鼠中,１０．５％出现清晰杂交信号。 表明：ｈＤＡＦ基因表达载体构建成功;并整合入小鼠基因组中。     

CRYOPRESERVATION OF SEA URCHIN EMBRYOS AND SPERM

A simple method for preserving live sea urchin embryos and sperm in liquid nitrogen (LN) wasdeveloped through the use of DMSO as a cryoprotective additive. Samples of late embryos in double test tubes were cooled to— I96°C by two-step freezing, first to — 76°C and then by plunging in LN. In the case of fertilized eggs, samples were previously frozen to — 40°C, at which temperature the samples were kept for 15 min; they were then transferred into LN. After preservation in LN for various lengths of time, samples in the double test tubes were thawed in water at 15°C. The post-thaw survival was more than 90% for late embryos, and about 10% for fertilized eggs. Difference in the length of the cryopreservation period did not affect survival. Post-thaw development in cryopreserved embryos often showed abnormalities in structure. Sperm with 1.5 M DMSO was successfully preserved in LN by a similar method to the one used for cryopreservation of late embryos. Fertilizability in cryopreserved sperm was complete, regardless of the length of the preservation period. Nearly all the eggs fertilized by cryopreserved sperm developed to normal plutei.     

钙蛋白酶抑制蛋白转基因小鼠的构建和鉴定

目的：将人的钙蛋白酶抑制蛋白（ calpastatin, CAST）外源基因整合到C57BL/6J小鼠中,构建高表达CAST的转基因小鼠模型。方法利用Gateway技术构建pRP．EX3d-EF1A-CAST-IRES-eGFP载体,回收片段后通过显微注射法将目的基因片段注入到C57 BL/6 J小鼠受精卵中,将其胚胎移植至同期发情的假孕受体母鼠输卵管内获得子代小鼠。采用PCR方法鉴定出阳性的转基因小鼠,确定首建鼠,通过与C57BL/6J小鼠回交后互交数代建系。利用RT-PCR和Western blotting方法检测CAST基因和蛋白在各组织中的表达情况。结果将90枚注射受精卵移植到3只假孕鼠中,3只均怀孕,移植成功率100％,产下23只子鼠,经PCR鉴定得到2只转基因阳性首建鼠,阳性率为9％。子代小鼠进行RT-PCR检查显示,CAST基因在转基因小鼠的心、肝、脾、肺、肾、脑和骨骼肌中均有表达;Western blotting检查显示,CAST蛋白表达在转基因小鼠中显著高于同窝阴性小鼠。结论通过显微注射法成功构建CAST高表达的转基因小鼠,为进一步研究CAST奠定了良好的模型基础。     

Production of transgenic rats using cryopreserved pronuclear‐stage zygotes

We investigated the application of cryopreserved pronuclearstage zygotes for the production of transgenic rats. Most of the pronuclearstage zygotes cryopreserved by conventional twostep freezing or vitrification appeared morphologically normal, but the proportion of frozen zygotes that developed into fetuses following transfer (59.7–60.2%) was higher than that of vitrified zygotes (5.5–22.1%). When the frozenthawed zygotes were used for DNA microinjection, 97.5% survived after DNA microinjection and 25.1% of the transferred zygotes developed into fetuses. These proportions were comparable to those of the fresh control zygotes (97.0% and 30.0%, respectively). The integration efficiency of the exogenous DNA into fetuses was similar between the frozen group (3.3% per injected zygote) and the control group (3.5%). These results indicate that pronuclearstage rat zygotes can be successfully cryopreserved by conventional twostep freezing for production of transgenic rats.     

Successful production of piglets derived from vitrified morulae and early blastocysts using a microdroplet method

This study was conducted to determine the efficiency of vitrification using the microdroplet (MD) method for early stage porcine embryos. Embryos at compacted morulae to early blastocyst stage were vitrified in a vitrification solution containing 40% (v/v) ethylene glycol, 0.6M sucrose and 2% (w/v) polyethylene glycol in M2 (ESP) without any pretreatment. The equilibration and dilution were carried out in third and fourth steps, respectively, at 38 degrees C. The survivability of the cryopreserved embryos was assessed for both in vitro culture (Experiment 1) and by embryo transfer (Experiment 2). In Experiment 1, the embryos were vitrified within a microdroplet or 0.25 ml straw (ST) and fresh embryos were used as a control group. The survival rates after 24h culture in the MD, ST and control groups were 21/23, 14/20 and 20/20, respectively. The hatching rates of the embryos after 48 h incubation were 14/23, 4/20 and 16/20, respectively. In Experiment 2, 171 vitrified embryos were transferred to 5 recipient gilts, and 17 healthy piglets were produced from 2 recipients (3 recipients aborted) in Group 1. In Group 2, 81 vitrified embryos and 16 fresh embryos in total were transferred to 4 recipient gilts, and 10 healthy piglets from the vitrified embryos were produced from 3 recipients. These results indicated that porcine embryos of compacted morulae to early blastocyst stage can survive cryopreservation using the microdroplet method without any special intracellular manipulation or treatment.     

Cryopreservation of spermatozoa of the American oyster, Crassostrea virginica Gmelin

Spermatozoa of the American oyster Crassostrea virginica were frozen to ?196 °C in concentrated Hanks' salt solution (2.6×) containing 8% dimethyl sulfoxide. About 0.2 ml of spermatozoa that were stored in liquid nitrogen for 68 days fertilized 91% of 65,600 eggs compared to fresh spermatozoa, which fertilized 92% of approximately the same number of eggs from the same females. Larvae resulting from fertilization of fresh eggs with 39-day-old cryopreserved spermatozoa appeared normal after 11 days.     

Live mice from cryopreserved embryos derived in vitro with cryopreserved ejaculated spermatozoa

This study was undertaken to try to reduce the number of animals required to maintain mouse strains by banking of embryos or spermatozoa. The principal objective was to cryopreserve ejaculated mouse spermatozoa, using a method recently developed for epididymal spermatozoa. Within 30 min after mating, ejaculated spermatozoa were flushed from the uterus of mated females; shortly afterwards, epididymal spermatozoa were also collected from the same males that had mated with the females. The average values for spermatozoal motility and viability of ejaculated specimens of nine males were 43 and 46%, respectively, and for epididymal specimens, the corresponding values were 60 and 52%. In experiment 1, ejaculated or epididymal spermatozoa were incubated with oocytes for 0.5 to 4 h. As evidenced by development into two-cell embryos within 24 h, kinetics of fertilization of the two spermatozoa types were similar. In experiment 2, ejaculated and epididymal spermatozoa of three males were separately cryopreserved in medium containing raffinose, glycerol, and egg yolk. Samples were cooled and seeded at -4 degrees C, cooled to -70 degrees C at 20 degrees C/min, and then were placed into liquid nitrogen for storage. When cryopreserved epididymal or ejaculated spermatozoa were thawed at > 1,000 degrees C/min and used for in vitro fertilization, > 60% of oocytes cleaved, and approximately 95% of cleaved embryos developed into morulae or blastocysts. When embryos produced with cryopreserved spermatozoa were transferred into recipients, 18 and 22 live pups were obtained from 62 and 54 embryos resulting from ejaculated or epididymal spermatozoa, respectively. This study documented the feasibility of cryopreserving ejaculated spermatozoa as an effective alternative to preserving germ plasm from genetically valuable mice.     

High-efficiency vitrification protocols for cryopreservation of in vitro grown shoot tips of transgenic papaya lines

In vitro grown shoot tips of transgenic papaya lines (Carica papaya L.) were successfully cryopreserved by vitrification. Shoot tips were excised from stock shoots that were preconditioned in vitro for 45–50-day-old and placed on hormone-free MS medium with 0.09 M sucrose. After loading for 60 min with a mixture of 2 M glycerol and 0.4 M sucrose at 25°C, shoot tips were dehydrated with a highly concentrated vitrification solution (PVS2) for 80 min at 0°C and plunged directly into liquid nitrogen. The regeneration rate was approximately 90% after 2 months post-thawing. Successfully vitrified and warmed shoot tips of three non-transgenic varieties and 13 transgenic lines resumed growth within 2 months and developed shoots in the absence of intermediate callus formation. Dehydration with PVS2 was important for the cryopreservation of transgenic papaya lines. This vitrification procedure for cryopreservation appears to be promising as a routine method for cryopreserving shoot tips of transgenic papaya line germplasm.