Efficient selection of transgenic mouse embryos using EGFP as a marker gene

We have established a reliable method that uses the EGFP (Enhanced Green Fluorescent Protein) gene as a marker for selecting transgenic embryos from preimplantation embryos. Embryos that were subjected to the pronuclear microinjection of the CMV/β‐actin/EGFP fusion gene were cultured in vitro until they developed into the morulae‐ or blastocyst‐stage. The expression of EGFP was easily observed by a fluorescent microscopy. There appeared to be no damage to the in vivo developmental ability of the embryos in response to the EGFP excitation light, which utilized an IB filter for a period of 30 min. Modified PCR analysis using Dpn I and Bal 31 digestion of the embryonic DNA showed that all of the embryos expressing EGFP in all their cells were transgenic, while more than half with mosaic expression of EGFP were not transgenic. Approximately 77% of pups born from the embryos that uniformly expressed the EGFP gene were transgenic, while 21.4% of pups from the embryos with mosaic expression were transgenics. The results showed that the use of EGFP as a marker is very useful and reliable for selecting transgenic embryos, and that it is important to transfer the embryos expressing EGFP in all their cells to obtain truly transgenic animals. Mol. Reprod. Dev. 54:43–48, 1999. © 1999 Wiley‐Liss, Inc.     

Production of transgenic mice by microinjection of DNA into vitrified pronucleate stage eggs

Vitrification is a technique for cryopreserving cells without crystallization due to elevation of the viscosity during the cooling process. We have developed a rapid and convenient mean of, cryopreserving mouse preimplantation embryos by vitrification using a solution (hereafter named DPS) consisting of 2.75m dimethylsulfoxide, 2.75m propylene glycol and 1.0m sucrose.In vitro fertilized pronucleate stage eggs were used because a large number of stage-matched eggs can be obtained at once. Only successfully fertilized eggs were collected and vitrified in DPS. After warming, two DNA constructs were injected into a total of 257 cryopreserved eggs, of which 175 (68%) survived the injection and were transferred into six recipients. All recipients became pregnant and gave birth to a total of 20 pups. When these DNA constructs were concomitantly injected into fresh eggs, 18% of eggs that were transferred developed into live pups, which was the same as the 18% figure for the cryopreserved eggs. With respect to transgenesis, 40% of the pups (8/20) developed from vitrified eggs were transgenic. In terms of the injected eggs that had been transferred, 4.5% of the 213 fresh eggs and 3.1% of the 112 vitrified eggs developed into transgenic mice. These results indicate that the efficiency of production of transgenic mice from vitrified eggs is comparable to that from fresh eggs.     

Offspring derived from intracytoplasmic injection of transgenic rat sperm

The objective of the present study was to produce rat offspring by intracytoplasmic sperm injection (ICSI) using a Piezo-driven micromanipulator. Transgenic male rats carrying a green fluorescent protein gene (GFP: homozygous) were used as sperm donors. The epididymal spermatozoa were suspended and sonicated in m-KRB medium and were frozen in the same medium at –20°C until use. When the sperm heads were aspirated into injection pipettes 7–10m in diameter and introduced into oocytes from the Wistar strain, no offspring resulted from the transfer of 59 eggs. In contrast, the sperm heads were hung on the tip of injection pipettes 2–4m in diameter and introduced into the oocytes, use of Piezo resulting in the production of 18 transgenic offspring carrying the GFP gene from 181 eggs transferred. The oocytes from the Sprague–Dawley strain also supported full-term development following ICSI with three offspring resulting from 163 transferred eggs. In an additional ICSI trial, spermatozoa from infertile transgenic rats carrying human lactalbumin with the thymidine kinase gene (LAC3: heterozygous) were used. The spermatozoa of the LAC3 transgenic rats appeared to be defective and immotile because of the expression of thymidine kinase in the testes, and no ICSI offspring resulted from 218 transferred eggs. These results suggest that ICSI is applicable in rats when Piezo-driven smaller pipettes are used to inject sperm heads together with a limited amount of the surrounding medium and that the ability of isolated sperm heads to participate in normal embryo development is maintained under the cryopreservation conditions employed.     

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.     

SHORT COMMUNICATION: Double pronuclei injection of DNA into zygotes increases yields of transgenic mouse lines

Transgenic mice are increasingly used for gene function and regulation studies of mammalian genes. A major limitation is the necessity to produce a large number of founder animals to obtain one line with the desired expression pattern. We developed a method, the 'double pronuclei injection', that doubles the yield of transgenic mouse lines obtained from each injection session, thereby reducing the time, effort and costs of generating transgenic mice. Three transgenic vectors were microinjected into the male and female pronuclei of zygotes. Approximately half of the resulting born mice were transgenic. This represented a 60% increase in the yield of founders per injected zygote, and a 100% increase in the yield of transgenic mice per born animal, when compared to yields obtained using single pronucleus injection. This method should prove useful for generating large numbers of transgenic mice for gene regulation studies and for conditional gene ablation     

Introduction of foreign DNA into walled plant cells via liposomes injected into the vacuole: a preliminary study

Plasmids containing the firefly luciferase reporter gene were introduced into tobacco and maize by electroporation. They were used to calibrate the performance characteristics of an Hamamatsu C1966 AVEC/VIM photonic camera-Leitz photomicroscope image processing system. Luciferin-dependent light emission was readily detected, on an individual cell basis, using the analytical photon counting mode of the Hamamatsu system. An efficient liposome-DNA encapsulation protocol was developed and used to introduce the firefly luciferase plasmid into walled cells of tobacco, maize, carrot and rice. This was achieved by pressure-injecting the liposomes (DNA encapsulated inside the vesicle) into the vacuole where they subsequently fused with the tonoplast, releasing the DNA into the cytoplasm. Analytical photon counting studies were conducted on injected cells to determine whether the DNA introduced in this way was expressed. To date all experiments have proved negative. Reasons for this lack of expression are discussed.     

Comparison between PMSG- and FSH-induced superovulation for the generation of transgenic rats

Superovulation protocols using single injections of pregnant mare's serum gonadotropin (PMSG) or minipumps with follicle-stimulating hormone (FSH) were compared in immature Sprague-Dawley (SD) rats. We used the following criteria: total number of ova, rate of fertilization, in vitro embryo development, sensitivity of zygotes to the microinjection of foreign DNA into the pronucleus, and their in-vivo development after transplantation into the oviduct of a recipient. Female SD rats were stimulated with 15 IU PMSG or 10 mg FSH followed by the injection of human chorionic gonadotropin (hCG) at doses of 20 and 30 IU per female. After hCG administration, they were mated with males of the same strain and sacrificed on day 1 of pregnancy. The percentage of mated animals and the fertilization rate was similar in all groups. In rats given PMSG, the number of ovulated zygotes was hCG dose-dependent. In contrast, the dose of hCG did not influence the efficiency of superovulation in rats given FSH, which was equal to PMSG-treated rats at the optimal dose of hCG. The rates of in vitro blastocyst development (31.4 and 23.3%) and the resistance to microinjection into the pronucleus did also not differ significantly between zygotes of both studied groups. The proportion of offspring developing from microinjected zygotes after oviduct transfer (26.2 and 26.8%, respectively) and the rate of transgene integration per newborns (7.3 and 4.9%, respectively) was similar in both experimental groups. The results of this study demonstrate that superovulation of immature SD rats by PMSG is equally effective as FSH treatment and, thus, preferable for transgenic rat technology due to the lower costs and easier handling.     

production of transgenic rodents by the microinjection of cloned DNA into fertilized one-cell eggs

Transgenic technologies that enable rapid movement between genotype and phenotype through specific loss-of-function, overexpression, or misexpression phenotypes will be crucial in the elucidation of gene sequences emerging from genome projects. This article describes detailed procedures for the generation of transgenic mice and rats by the injection of cloned DNA into the pronuclei of fertilized one-cell eggs.     

Production of transgenic blastocyst by nuclear transfer from different types of somatic cells in cattle

The present study examined the effects of genetic manipulation to the donor cell and different types of transgenic donor cells on developmental potential of bovine nuclear transfer (NT) embryos. Four types of bovine somatic cells, including granulosa cells, fetal fibroblasts, fetal oviduct epithelial cells and fetal ovary epithelial cells, were transfected with a plasmid (pCE-EGFP-Ires-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes by electroporation. After 14 days selection with 800 μg/mL G418, transgenic cell lines from each type of somatic cells were obtained. Nontransgenic granulosa cells and all 4 types of transgenic somatic cells were used as nuclear donor to produce transgenic embryos by NT. There was no significant difference in development rates to the blastocyst stage for NT embryos from transgenic and nontransgenic granulosa cells (44.6% and 42.8%, respectively), and transfer of NT embryos derived from transgenic and nontransgenic granulosa cells to recipients resulted in similar pregnancy rates on day 90 (19% and 25%, respectively). The development rates to the blastocyst stage of NT embryos were significantly different among different types of transgenic donor cells (P<0.05). Blastocyst rates from fetal oviduct epithelial cell and granulosa cell (49.1% and 44.6%, respectively) were higher than those from fetal fibroblast (32.7%) and fetal ovary epithelial cell (22.5%). These results suggest that (i) genetic manipulation to donor cells has no negative effect on in vitro and early in vivo developmental competence of bovine NT embryos and (ii) granulosa and fetal oviduct epithelial cells can be used to produce transgenic bovine NT embryos more efficiently. In addition, GFP can be used to select transgenic NT embryos as a non-invasive selective marker.     

Production of transgenic blastocyst by nuclear transfer from different types of somatic cells in cattle

Genetically modified animals have many poten-tial applications in basic research, human medicine and agriculture. Pronuclear DNA microinjection has been almost the only practical means of producing transgenic animals during the last 20 years, but the low efficiency (1%—5%)[1] of this method has actu-ally been the obstacle that hampered its further appli-cation in animal biotechnology. The birth of Dolly[2], the first somatically cloned animal, made it possible to produce transgenic animals b…     

Transgenesis in mice by cytoplasmic injection of polylysine/DNA mixtures

Pronuclear injection is currently the most often used method to make transgenic animals, but in some animal species it is temporally restrictive due to difficulty in visualizing pronuclei. However, the injection of construct DNA into the cytoplasm does not result in transgenesis. The production of transgenic mice by a cytoplasmic microinjection technique of polylysine complexed DNA into pronuclear stage zygotes is described. Transgenic mice were produced from cytoplasmic microinjection of mixtures of a 5.3 kb linearized DNA and poly-l-lysine (degree of polymerization=51). Effects on transgenic frequency of both the lysine to phosphate ratio of polylysine to DNA and DNA concentration were studied. About 12.8% of the pups born from zygotes cytoplasmically microinjected with a polylysine/DNA mixture having a lysine to phosphate ratio (L:P) of 11 microinjection positive control of DNA alone was 21.7%. No transgenic pups were born from microinjection of DNA alone into the cytoplasm. Complexes of polylysine/DNA were detected using agarose gel electrophoresis at the conditions which produced transgenic mice. The presence of polylysine with construct DNA altered thein vitro activities of restriction endonuclease and DNA ligase on the construct DNA. The production of transgenic animals using DNA and polylysine in the absence of any other signal protein suggests that a DNA/polylysine complex but not DNA alone can act as a substrate for transgenesis from the cytoplasm.     

Rescue of infertile transgenic rat lines by intracytoplasmic injection of cryopreserved round spermatids

Transgenic male rats carrying human alpha-lactalbumin with thymidine kinase gene (line name; LAC3) were found to be infertile due to expression of the transgene in the testes. Furthermore, it was not possible to maintain the line even by the use of intracytoplasmic sperm injection (ICSI). Therefore, round spermatids prepared from the LAC3 rats were microinjected into strontium-activated oocytes using a Piezo-driven micromanipulator. Of 263 oocytes microinjected with LAC3 spermatids, 244 (92.8%) survived the injection and 96 (39.3%) developed to the 2-cell stage. Three viable offspring were born after transfer (1.4%, 3/219), and two offspring carried the LAC3 transgene. In the control experiment using spermatids of Wistar rats, similar proportions of post-injection survival (91.3%, 241/264), cleavage (40.2%, 97/241), and development into offspring (0.5%, 1/206) were obtained. Thus, this paper reports not only the first rat offspring derived from round spermatid injection but also the practical application of the microinsemination technique to the rescue of transgenes of infertile transgenic male rats.     

Factors affecting production of transgenic rats by ICSI-mediated DNA transfer: effects of sonication and freeze-thawing of spermatozoa, rat strains for sperm and oocyte donors, and different constructs of exogenous DNA

Factors affecting the efficiency of producing transgenic rats by intracytoplasmic sperm injection (ICSI)-mediated DNA transfer were investigated. Epididymal spermatozoa from Sprague-Dawley (SD) rats were sonicated and/or frozen-thawed for cutting the tail and membrane disruption. The sperm heads were exposed for 1 min to different concentrations (0.02-2.5 microg/ml) of 3.0 kb enhanced green fluorescent protein (EGFP) DNA solution, and then microinjected into the denuded F1 hybrid (Donryu x LEW) rat oocytes. The optimal concentration of EGFP DNA solution was 0.1 microg/ml, as determined by the in vitro developmental competence into morulae/blastocysts of the ICSI oocytes and the EGFP expression of the resultant embryos. The efficiency of producing transgenic rat offspring (per transferred zygote) was 2.8%, 1.6%, and 3.3% in the oocytes injected with sonicated, frozen-thawed, and sonicated + frozen-thawed sperm heads, respectively. The founder transgenic rats carrying the EGFP gene transmitted their transgenes to their progeny according to the Mendelian fashion, suggesting the stable incorporation of the transgenes into the rat genomes. Four rat strains (F344, LEW, Donryu, and SD) were compared for their suitability as sperm/oocyte donors for the production of transgenic rats by ICSI with sonicated, frozen-thawed and solution of EGFP DNA-exposed sperm heads. The efficiency of producing transgenic rats in the SD strain (8.2%) was higher than that in the LEW strain (0.9%), while those in the F344 and Donryu strains (4.3%-4.4%) were intermediate. One plasmid DNA (Fyn, 5.0 kb) and two BAC DNA (BAC/Fyn, 208 kb; Svet1/IRES-Cre, 186 kb) were successfully introduced into the SD rat genomes via ICSI, with the producing efficiencies of 2.8%, 0.9%, and 2.4%, respectively.     

Production of a transgenic piglet by a sperm injection technique in which no chemical or physical treatments were used for oocytes or sperm

As a method of producing transgenic animals, spermatozoa have been used to fertilize mammalian oocytes through natural copulation, artificial insemination (AI), and in vitro fertilization (IVF). Our objective was to produce live piglets expressing the enhanced green fluorescent protein (eGFP) by the modified ICSI procedure based on Yong et al. (2003) (Hum. Reprod. 18:2390) where this procedure resulted in an improvement in development in vitro as compared to conventional ICSI and IVF. After injecting frozen-thawed sperm, recovered from the descendant of a transgenic boar derived by oocyte transduction, into in vitro matured oocytes the injected oocytes were surgically transferred into the oviduct of six surrogate gilts. Two gilts (33%) became pregnant. One gave birth to a healthy male piglet. Expression of the eGFP was easily observed in the nose and hooves by direct epifluorescent examination in the newborn piglet. These results show the production of the first viable transgenic piglet by in vitro maturation and our new sperm injection method.     

Production of inbred and hybrid transgenic mice carrying large (> 200 kb) foreign DNA fragments by intracytoplasmic sperm injection

We have developed a mouse transgenesis technique that facilitates the insertion of large (approximately 200 kilo base pairs) DNA fragments into host genomes of both inbred and hybrid mice. Six inbred and three hybrid transgenic mice carrying a single bacterial artificial chromosome (BAC) clone with genes located in the Down syndrome critical region of human chromosome 21 were produced using this technology.     

Efficient production of intersubspecific hybrid mice and embryonic stem cells by intracytoplasmic sperm injection

Recently, mice and embryonic stem (ES) cells with allelic polymorphisms have been used extensively in the field of genetics and developmental biology. In this study, we examined whether intersubspecific hybrid mice and ES cells with these genotypes can be efficiently produced by intracytoplasmic sperm injection (ICSI). Frozen-thawed spermatozoa from wild-derived strains, JF1 (Mus musculus molossinus), MSM (M. m. molossinus), HMI (M. m. castaneus), and SWN (M. m. spp.), were directly injected into mature oocytes from laboratory mice ([C57BL/6 x DBA2]F1; M. m. domesticus). The in vitro and in vivo developmental capacity of F1 embryos was not significantly different among the groups (P > 0.05), and term offspring were efficiently obtained in all groups (27%-34% of transferred embryos). However, the mean body and placental weights of the offspring differed significantly with genotype (P < 5 x 10(-10)), with the HMI hybrid greatest in both body and placental weights. In an application study using these F1 offspring, we analyzed their mitochondrial DNA using intersubspecific polymorphisms and found the consistent disappearance of sperm mitochondrial DNA in the F1 progeny. In a second series of experiments, we generated F1 blastocysts by injecting MSM spermatozoa into C57BL/6 oocytes and used them to generate hybrid ES cell lines. The ES cell lines were established at a high efficiency (9 lines from 20 blastocysts) and their allelic polymorphisms were confirmed. Thus, ICSI using cryopreserved spermatozoa allows the efficient and immediate production of a number of F1 hybrid mice and ES cell lines, which can be used for polymorphic analysis of mouse genetics.     

Assessment of DNA damages in lymphocytes of agricultural workers exposed to pesticides by comet assay in a cross-sectional study

Purpose: To assess the predictive power of the comet assay in the context of occupational exposure to pesticides.

Materials and methods: The recruited subjects completed a structured questionnaire and gave a blood sample. Exposure to pesticides was measured by means of an algorithm based on Dosemeci’s work (Agricultural Health Study). Approximately 50 images were analyzed for each sample via fluorescence microscopy. The extent of DNA damage was estimated by tail moment (TM) and is the product of tail DNA (%) and tail Length.

Results: Crude significant risks (odds ratios, ORs) for values higher than the 75th percentile of TM were observed among the exposed subjects (score?>?1). The frequency of some confounding factors (sex, age and smoking) was significantly higher among the exposed workers. A significant dose–effect relationship was observed between TM and exposure score. Significant high-risk estimates (ORs), adjusted by the studied confounding factors, among exposure to pesticides and TM, % tail DNA and tail length were confirmed using unconditional logistic regression models.

Conclusions: The adjusted associations (ORs) between the comet parameters and exposure to pesticides were significant. The sensitivity of the comet test was low (41%), the specificity (89%) and the predictive positive value (0.77) were found acceptable.     

Position-independent and high-level expression of human α-lactalbumin in the milk of transgenic rats carrying a 210-kb YAC DNA

The level of expression of transgenes in transgenic animals varies among lines, and is often much lower than that of endogenous genes (position effects). In order to surmount position effects and establish a more efficient production system of transgenic animals producing pharmaceutical proteins in their milk, transgenic rats carrying 210-kb YAC DNA containing the human α-lactalbumin gene were produced. Three transgenic lines transmitted the transgene to the next generation. They had one copy of the α-lactalbumin gene and secreted human α-lactalbumin in their milk at concentrations of 2.0–4.3 mg/ml. No position effect was seen. The transgene was expressed specifically in the mammary gland of the transgenic rats. The 210-kb region is thought to contain all the DNA elements required for proper expression of the human α-lactalbumin gene. The YAC carrying the human α-lactalbumin gene is a potential vector for the expression of foreign genes in the mammary gland. Mol. Reprod. Dev. 47:157–163, 1997. © 1997 Wiley-Liss, Inc.