Pronuclear visibility, development and transgene expression in IVM/IVF-derived porcine embryos

A total of 1550 zygotes was used to assess the timing of pronuclear visibility, embryo development following DNA microinjection, and transgene expression in IVM/IVF-generated porcine embryos. After centrifugation, pronuclei could be seen in 61.6% of zygotes. In 55.3% of these only 1 pronucleus was visible. Pronuclear visibility was highest at 20 h post-insemination. Zygotes were microinjected with 1 of 2 LacZ gene constructs driven by either the SV40 early promoter (pSVON) or the human cytoplasmic beta actin promoter (pbActinLacZ). Development and transgene expression were assessed after either 48 h or 7 d in culture. After 48 h, significantly more zygotes with a single visible pronucleus developed to the 8-cell stage than zygotes in which no pronucleus had been seen (43.0 vs 24.8%), while those with 2 pronuclei were intermediate (31.4%). After 7 d, no difference in development to the morula stage was observed between noninjected control embryos (25.5%) and embryos with 1 (21.0%) or 2 pronuclei (22.5%); however, the proportion of embryos reaching the morula stage in the nonpronuclear group was significantly reduced (9.1%). After 48 h in culture, transgene expression was significantly higher in embryos with 2 pronuclei at the time of injection than in those with 1 (36.4 vs 17.9%). After 7 d in culture, 41.5% of morulae derived from zygotes with 2 pronuclei and 29.97% of thsoe derived from zygotes with 1 pronucleus showed signs of transgene expression. At this stage, significantly more morulae expressed the pbActinLacZ than the pSVON transgene (43.8 vs 25.8%). More than 80% of putative transgenic morulae or blastocysts showed evidence of mosaicism. These results demonstrate that IVM/IVF porcine embryos are able to develop in culture and express a microinjected transgene.     

Genomic DNA damage in mouse transgenesis

Creating transgenic mammals is currently a very inefficient process. In addition to problems with transgene integration and unpredictable expression patterns of the inserted gene, embryo loss occurs at various developmental stages. In the present study, we demonstrate that this loss is due to chromosomal damage. We examined the integrity of chromosomes in embryos produced by microinjection of pronuclei, intracytoplasmic sperm injection (ICSI), and in vitro fertilization (IVF)-mediated transgenesis, and correlated these findings with the abilities of embryos to develop in vitro and yield transgenic morulas/blastocysts. Chromosomal analysis was performed after microinjection of the pronuclei in zygotes, as well as in parthenogenetic and androgenetic embryos. In all the pronuclei injection groups, significant oocyte arrest and increased incidence of chromosome breaks were observed after both transgenic DNA injection and sham injection. This indicates that the DNA damage is a transgene-independent effect. In ICSI-mediated transgenesis, there was no significant oocyte arrest. The observed chromosomal damage was lower than that after pronuclei microinjection in zygotes and was dependent upon the presence of exogenous DNA. The occurrence of DNA breaks, as measured by comet assay performed on the sperm prior to ICSI, showed that DNA damage was present in the sperm before fertilization. Embryonic development in vitro and transgene expression at the morula/blastocyst stage were higher in ICSI-mediated transgenesis than after microinjection of pronuclei into zygotes. Sperm-mediated gene transfer via IVF did not affect chromosome integrity, allowed good embryo development, but did not yield any transgenic embryos. The present study demonstrates that DNA damage occurs after both the microinjection of pronuclei and ICSI-mediated transgenesis, albeit through different mechanisms.     

Characterization of the ICSI-mediated gene transfer method in the production of transgenic pigs

Understanding the behavior of transgenes introduced into oocytes or embryos is essential for evaluating the methodologies for transgenic animal production. We investigated the expression pattern of a transgene transferred to porcine eggs by intracytoplasmic sperm injection‐mediated gene transfer (ICSI‐MGT) or pronuclear microinjection (PN injection). The introduction of the EGFP gene by ICSI‐MGT yielded significantly more embryos with non‐mosaic transgene expression (P < 0.01). In the ICSI‐MGT group, 61.5% (24/39) of the embryos were EGFP‐positive in all their component blastomeres at the morula stage, while fewer than 10% of such embryos were EGFP‐positive in the PN‐injection group. Using three types of transgenes, ranging from 3.0 to 7.5 kb in size, we confirmed that approximately one in four fetuses obtained by ICSI‐MGT was transgenic, suggesting that ICSI‐MGT is a practical method for transgenic pig production. Southern blot analysis of 12 transgenic fetuses produced by ICSI‐MGT revealed that the number of integrated transgene copies varied from 1 to 300, with no correlation between transgene size and the number of integrated copies. Fluorescence in situ hybridization analysis revealed that the transgenes were randomly integrated into a single site on the host chromosomes. Together, these data indicate that multiple‐copy, single‐site integration of a transgene is the primary outcome of ICSI‐MGT in the pig and that ICSI‐MGT is less likely than PN injection to cause transgene integration in a mosaic manner. Mol. Reprod. Dev. 79: 218–228, 2012. © 2011 Wiley Periodicals, Inc.     

Timing of DNA integration, transgenic mosaicism, and pronuclear microinjection

Selection of transgenic embryos prior to embryo transfer is a means to increase the efficiency of transgenic livestock production. Among transgenic reporters, cytoplasmic expression of green fluorescent protein (GFP) has features that make it ideal for transgenic embryo selection. The primary objective of this study was to assess cytoplasmic expression of a specially designed GFP reporter as a tool for transgenic bovine embryo selection. A second objective was to evaluate this reporter for studying transgenic mosaicism related to timing of integration of pronuclear microinjected DNA. Transgenic embryos produced by pronuclear injection showed a discrete pattern of GFP expression with clusters at 25, 50, and 100% of blastomeres expressing GFP. This pattern of mosaicism is interpreted to indicate that the integration of microinjected DNA occurred, not only at the pronuclear stage, but also in the subsequent cell divisions. Among the GFP-positive transgenic embryos, only in 21% did all the blastomeres show the green fluorescence. Using the fraction of positive blastomeres within an embryo, the timing of integration of microinjected DNA was estimated. The frequency of nonmosaic embryos expressing GFP is consistent with published germline transmission success rates of transgenic cattle derived from pronuclear microinjected embryos. These results indicate the possible application of GFP as a marker of transgenic embryos and graphically illustrate underlying complexities in DNA integration in embryos subjected to pronuclear microinjection.     

Production of functional transgenic mice by DNA pronuclear microinjection

Successful experiments involving the production of transgenic mice by pronuclear microinjection are currently limited by low efficiency of random transgene integration into the mouse genome. Furthermore, not all transgenic mice express integrated transgenes, or in other words are effective as functional transgenic mice expressing the desired product of the transgene, thus allowing accomplishment of the ultimate experimental goal - in vivo analysis of the function of the gene or gene network. The purpose of this review is to look at the current state of transgenic technology, utilizing a pronuclear microinjection method as the most accepted way of gene transfer into the mouse genome.     

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.     

The Meganuclease I-SceI Containing Nuclear Localization Signal (NLS-I-SceI) Efficiently Mediated Mammalian Germline Transgenesis via Embryo Cytoplasmic Microinjection

The meganuclease I-SceI has been effectively used to facilitate transgenesis in fish eggs for nearly a decade. I-SceI-mediated transgenesis is simply via embryo cytoplasmic microinjection and only involves plasmid vectors containing I-SceI recognition sequences, therefore regarding the transgenesis process and application of resulted transgenic organisms, I-SceI-mediated transgenesis is of minimal bio-safety concerns. However, currently no transgenic mammals derived from I-SceI-mediated transgenesis have been reported. In this work, we found that the native I-SceI molecule was not capable of facilitating transgenesis in mammalian embryos via cytoplasmic microinjection as it did in fish eggs. In contrast, the I-SceI molecule containing mammalian nuclear localization signal (NLS-I-SceI) was shown to be capable of transferring DNA fragments from cytoplasm into nuclear in porcine embryos, and cytoplasmic microinjection with NLS-I-SceI mRNA and circular I-SceI recognition sequence-containing transgene plasmids resulted in transgene expression in both mouse and porcine embryos. Besides, transfer of the cytoplasmically microinjected mouse and porcine embryos into synchronized recipient females both efficiently resulted in transgenic founders with germline transmission competence. These results provided a novel method to facilitate mammalian transgenesis using I-SceI, and using the NLS-I-SceI molecule, a simple, efficient and species-neutral transgenesis technology based on embryo cytoplasmic microinjection with minimal bio-safety concerns can be established for mammalian species. As far as we know, this is the first report for transgenic mammals derived from I-SceI-mediated transgenesis via embryo cytoplasmic microinjection.     

Generation of transgenic goats by pronuclear microinjection: a retrospective analysis of a commercial operation (1995–2012)

Production of transgenic founder goats involves introducing and stably integrating an engineered piece of DNA into the genome of the animal. At LFB USA, the ultimate use of these transgenic goats is for the production of recombinant human protein therapeutics in the milk of these dairy animals. The transgene or construct typically links a milk protein specific promoter sequence, the coding sequence for the gene of interest, and the necessary downstream regulatory sequences thereby directing expression of the recombinant protein in the milk during the lactation period. Over the time period indicated (1995–2012), pronuclear microinjection was used in a number of programs to insert transgenes into 18,120, 1- or 2- cell stage fertilized embryos. These embryos were transferred into 4180 synchronized recipient females with 1934 (47%) recipients becoming pregnant, 2594 offspring generated, and a 109 (4.2%) of those offspring determined to be transgenic. Even with new and improving genome editing tools now available, pronuclear microinjection is still the predominant and proven technology used in this commercial setting supporting regulatory filings and market authorizations when producing founder transgenic animals with large transgenes (> 10 kb) such as those necessary for directing monoclonal antibody production in milk.     

A simple and highly efficient transgenesis method in mice with the Tol2 transposon system and cytoplasmic microinjection

Creating transgenic mice is an important technology for genetic studies and is currently performed by pronuclear microinjection of plasmid DNA into fertilized eggs. Since survival of injected embryos and integration of plasmid DNA are not efficient, total efficiency is only around 3% with a standard protocol. To circumvent this problem, here we describe a novel transgenesis method, the Tol2-mediated cytoplasmic injection method (Tol2:CI). We injected a foreign DNA cloned in a Tol2-transposon vector together with the transposase mRNA into the cytoplasm of fertilized eggs. As expected, the survival rate of the injected embryos was increased drastically. Also, the foreign DNA was transposed from the plasmid to the genome and transmitted to the next generation very efficiently. Together, the overall transgenic efficiency became more than 20%. Considering its simplicity and perfect compatibility with existing pronuclear microinjection facilities, we propose that the Tol2:CI method is applicable to high throughput functional genomics studies.     

Birth of normal kids after microinjection of pronuclear embryos in a transgenic goat (Capra hircus) production program in Brazil

This pilot project was designed to determine if normal kids could be produced after microinjection in pronuclear embryos and subsequent transfer to recipients in a transgenic goat program in Brazil. Twelve donors of the Saanen breed and 17 recipients of an undefined breed were used. The estrus of both donors and recipients was synchronized by a standard progestagen treatment and superovulation obtained by six pFSH injections. Donors in estrus were mated with fertile Saanen bucks. Zygotes were recovered surgically by flushing oviducts. The recovered zygotes with visible pronuclei were microinjected with 500 to 1000 copies of the human G-CSF gene. Two or four embryos were surgically transferred into the oviducts of recipients. One recipient became pregnant and two kids were born. No transgenic goat was identified after PCR analysis. Even though transgenic goats were not obtained, this experiment establishes the basis of a synchronization and superovulation regimen for use in goats raised in Brazil, for the purpose of collecting and manipulating the pronuclear embryos. This project also showed that microinjected one-cell goat embryos can survive to produce live young following surgical transfer.     

Episomal maintenance of a bovine papilloma virus vector in transgenic mice.

We have used a bovine papillomavirus-based vector to generate transgenic mice. Transgenic mice result from either pronuclear or cytoplasmic injections of the vector into fertilized eggs. Of 30 mice generated by microinjection, 27 (90%) contained the vector in its episomal form, at less than one copy per cell. This represents a highly efficient means of gene transfer in which the transgene is in a controlled genetic environment.     

Improving the generation of genomic-type transgenic mice by ICSI

Transgenes included in genomic-type constructs, such as yeast artificial chromosomes (YAC), P1-derived artificial chromosomes, or bacterial artificial chromosomes (BAC), are normally correctly expressed, according to the endogenous expression pattern of the homologous locus, because their large size usually ensures the inclusion of all regulatory elements required for proper gene expression. The use of these large genomic-type transgenes is therefore the method of choice to overcome most position effects, commonly associated with standard-type transgenes, and to guarantee the faithful transgene expression. However, in spite of the different methods available, including pronuclear microinjection and the use of embryonic stem cells as vehicles for genomic transgenes, the generation of transgenic animals with BACs and, particularly, with YACs can be demanding, because of the low efficiencies requiring extensive microinjection sessions and/or higher number of oocytes. Recently, we have explored the use of intracytoplasmic sperm injection (ICSI) into metaphase II oocytes as an alternative method for the generation of YAC transgenic mice. Our results suggest that the use of transgenic strategies based on ICSI significantly enhances the efficiency of YAC transgenesis by at least one order of magnitude.     

Kinetics of pronuclear development and the effects of vector type and timing of injection on the efficiency of gene transfer into rhesus macaque embryos

A series of experiments was performed to determine the dynamics of pronuclear development as well as the efficiency of either adenovirus-associated (AAV) or lentivirus-derived vectors to introduce a green fluorescent protein (GFP) reporter gene into rhesus macaque (Macaca mulatta) embryos. Assessment of pronuclear development at various times after fertilization revealed that the appearance of pronuclei was determined by the presence of the first and the timing of the second polar body. The dynamics of pronuclear formation was a significant determinant of whether an oocyte reached the blastocyst stage, however, when the percentage of blastocysts were based on the number of zygotes, the timing of the appearance of polar bodies did not appear to have any effect on subsequent development. Injection of different AAV-derived vectors showed that the serotype of the vector did not affect development or the proportion of transgenic embryos. Moreover, all putative transgenic embryos proved to be expression mosaics. Injection of embryos with lentiviral vectors showed that timing of injection (before or after fertilization) had no effect on subsequent transgene expression, but that the type of reporter gene determined post-injection development and rate of transgenesis. The transfer of embryos following injection of a lentiviral vector into three recipients resulted in one pregnancy which was lost during the second trimester. Analysis of fetal tissues showed ubiquitous presence of the transgene and GFP expression in all tissues examined. These results show that lentivirus-derived vectors can efficiently transform rhesus embryos and are suitable for the generation of transgenic rhesus monkeys.     

Increased transgene integration efficiency upon microinjection of DNA into both pronuclei of rabbit embryos

Transgenic rabbits provide a useful biological model for the study of the regulation of mammalian genes. However, transgene integration efficiency has generally been low. Here we present a first attempt to increase the integration rate of exogenous DNA into the rabbit genome, using a double pronuclei microinjection method. Pronuclear stage rabbit embryos were recovered from superovulated NZW females, 19–20 h after hCG injection. About 5 μg/mL of exogenous DNA solution was microinjected either into one pronucleus (single microinjection, SM) or into both pronuclei (double microinjected, DM). The transgene consisted of a 2.5 kb murine whey acidic protein promoter (mWAP), 7.2 kb cDNA of the human clotting factor VIII (hFVIII), and 4.6 kb that of 3′ flanking sequences of the mWAP gene. The in vitro survival of DM embryos to the blastocyst stage was lower than that of SM embryos (68 vs. 89%). Similar results were obtained using EGFP as a control gene construct. However, there was no difference in the percentage of embryos that developed into live offspring using DM (25%) vs. SM (26%). The integration frequency of mWAP-hFVIII into the genome of transgenic rabbits was 3.3% (1/30) upon SM and 8.1% (4/49) at DM (p < 0.05). All founders transmitted the transgene to their offspring in a Mendelian fashion. The SM founder female secreted 87.4 μg/mL rhFVIII in milk, with an activity of 0.594 lU/mL. The DM founder female produced 118 μg/mL rhFVIII, with activity values of 18 lU/mL. This is the first report of transgenic rabbit production using a double microinjection technique. Our preliminary results suggest that this method can increase the efficiency of production of transgenic rabbit founders, giving a higher integration rate than single microinjection.     

Prediction of Transgene integration by Noninvasive Bioluminescent Screening of Microinjected Bovine Embryos

Transgenesis in domestic species, as a research tool and in biotechnological applications, has been limited by the expense of producing transgenic offspring by standard microinjection techniques. A major factor is the inefficiency of maintaining large numbers of recipient females, when a high percentage of these carry nontransgenic fetuses. There are two approaches to reduce this cost, the fusion of transfected fetal fibroblasts with enucleated oocytes, and the screening of microinjected embryos for transgene integration in blastocysts, prior to transfer. Here, we develop a luminescent screening system to select transgenic bovine embryos. A transgene with scaffold attachment regions flanking the murine HSP70.1 promoter linked to firefly luciferase cDNA, was microinjected into pronuclei of in vitro produced zygotes. At the blastocyst stage, the transgene was induced by heat shock (45 °C, 15 min) and 4–6 h later, luciferase expression was analyzed by photon counting imaging. Screened blastocysts were transferred to recipients and day 50 fetuses or calves were analyzed by PCR and Southern blot for transgene integration. When nonluminescent blastocysts were transferred, transgene integration was never observed. Of 13 fetuses derived from luminescent blastocysts, 3 contained integrated transgenes that were functional in all tissues examined. Image analysis of the signal emitted by positive blastocysts revealed that 9 nontransgenic fetuses were obtained from blastocysts that exhibited a localized luminescent signal. On the other hand, 3 of 4 fetuses derived from blastocysts that emitted light over more than 70% of their surface were transgenic. Thus, by selecting luminescent blastocysts on the basis of both signal intensity and distribution, the number of recipient females required to produce transgenic offspring can be greatly reduced. Using this technique it should also be possible to improve the efficiency of transgenesis by microinjection through studies in which vector design and integration conditions are examined at the blastocyst stage.     

Efficient generation of transgenic pigs using equine infectious anaemia virus (EIAV) derived vector

Traditional methods of transgene delivery in livestock are inefficient. Recently, human immunodeficiency virus (HIV-1) based lentiviral vectors have been shown to offer an efficient transgene delivery system. We now extend this method by demonstrating efficient generation of transgenic pigs using an equine infectious anaemia virus derived vector. We used this vector to deliver a green fluorescent protein expressing transgene; 31% of injected/transferred eggs resulted in a transgenic founder animal and 95% of founder animals displayed green fluorescence. This compares favourably with results using HIV-1 based vectors, and is substantially more efficient than the standard pronuclear microinjection method, indicating that lentiviral transgene delivery may be a general tool with which to efficiently generate transgenic mammals.     

Evaluating birth frequencies and embryonic lethality in transgenesis following pronuclear injection of HIV DNA

The likelihood that expression of a foreign gene in a mammalian cell is deleterious to viability is confronted whenever novel transgenic animals are made. A pathological response to transgene expression is even desired in transgenic mouse models of human disease. The derivation of HIV-transgenic mice in our laboratory using multiple recombinant forms of an HIV provirus has resulted in mixed success best explained by the variable toxicity of the different transgenes. Employing a standardized approach to pronuclear injections, experimental variation amongst recombinant HIV transgenes was documented in terms of the percentage of pregnancies following embryo transfer into pseudopregnant mice and the percentage of transplanted embryos leading to term births in these pregnant females (giving rise to an index of birth success, SI). Results compiled over 5 years suggested that the SI reflected transgene toxicity, in this case of HIV gene products early in embryogenesis. These observations have guided the design of productive transgenes for mouse models of HIV-related diseases and may be generally applicable in transgenesis.     

Relevance of BAC transgene copy number in mice: transgene copy number variation across multiple transgenic lines and correlations with transgene integrity and expression

Bacterial artificial chromosomes (BACs) are excellent tools for manipulating large DNA fragments and, as a result, are increasingly utilized to engineer transgenic mice by pronuclear injection. The demand for BAC transgenic mice underscores the need for careful inspection of BAC integrity and fidelity following transgenesis, which may be crucial for interpreting transgene function. Thus, it is imperative that reliable methods for assessing these parameters are available. However, there are limited data regarding whether BAC transgenes routinely integrate in the mouse genome as intact molecules, how BAC transgenes behave as they are passed through the germline across successive generations, and how variation in BAC transgene copy number relates to transgene expression. To address these questions, we used TaqMan real-time PCR to estimate BAC transgene copy number in BAC transgenic embryos and lines. Here we demonstrate the reproducibility of copy number quantification with this method and describe the variation in copy number across independent transgenic lines. In addition, polymorphic marker analysis suggests that the majority of BAC transgenic lines contain intact molecules. Notably, all lines containing multiple BAC copies also contain all BAC-specific markers. Three of 23 founders analyzed contained BAC transgenes integrated into more than one genomic location. Finally, we show increased BAC transgene copy number correlates with increased BAC transgene expression. In sum, our efforts have provided a reliable method for assaying BAC transgene integrity and fidelity, and data that should be useful for researchers using BACs as transgenic vectors.     

Transgenic fish

Transgenic fish are produced by the artificial transfer of rearranged genes into newly fertilized eggs. Currently microinjection is the preferred method, although the integration rates of transgenes are generally low. A number of fusion genes, containing retrovirus sequences which direct integration, have been developed to enhance integration of transgenes. Mass gene transfer methods are also being developed. These include lipofection, particle bombardment, and electroporation of embryos and sperm cells. These methods are potentially useful for marine organisms such as crustaceans and molluscs as well as fish. In contrast to microinjection, which treats single cells individually, these methods can transfer genes into a large number of eggs at once. There is some evidence to indicate successful integration and expression of transgenes transferred by the electroporation of embryos and sperm cells. Germline transmission of transgenes has been observed through mating studies, and in some cases the progeny express the new phenotype consistently. However, germline transmission does not necessarily confirm stable integration of the transgene. There is evidence that transgenes may exist extrachromosomally. Transgenic fish are viewed as a useful model for the study of complex biological phenomena such as growth and differentiation, and as a fast track to the production of broodstock for the aquaculture industry. Current research focuses on the elucidation of the mechanisms controlling the regulation of gene expression. The use of transgenic fish for the isolation of developmental genes has just begun. Applications of transgenesis to broodstock development have been focused on the development of fish with accelerated growth, tolerance to low temperature, and disease resistance. However, before the release of transgenic fish into the environment, the possible impact on the environment must be assessed. There must be safeguards to protect the genetic diversities of the natural populations, and to conserve the natural habitats     

Efficiency of transgenic rat production is independent of transgene-construct and overnight embryo culture

The aim of the present work was to study factors affecting the efficiency of transgenic technology in rats. We investigated the possible effects of pronuclear microinjection of buffer or different DNA-constructs on survival and development of rat zygotes in vitro and in vivo as well as the influence of overnight culture of these embryos before transfer into pseudopregnant foster mothers. The survival rate of zygotes and their development to the two-cell and morula stage was not affected by pronuclear microinjection with different DNA-constructs or buffer. However, the development to the blastocyst stage was impaired. Nevertheless, there was no difference in blastocyst development between zygotes injected with DNA-constructs or with buffer. Neither was there a difference in cell number in in vitro cultured blastocysts resulting from pronuclear microinjection of a transgene compared with non-injected controls. The survival rate to term was about 30% irrespective of whether microinjected embryos were transferred immediately after microinjection or after overnight culture in vitro. However, a reduction in the survival to term was observed for non-injected zygotes when they were developed in vitro to the two-cell stage before transfer to a pseudopregnant female. The percentage of transgenic rats that resulted from microinjected zygotes was similar in all groups regardless of the DNA-construct used (2.7-10.0%). In conclusion, the main detrimental factor in the microinjection of rat zygotes is the introduction of solution in the pronucleus. Overnight culture of zygotes between microinjection and oviduct transfer does not decrease the efficiency of transgenic rat generation.