Efficiency Of Transgene Integration Research Articles

A Nonhomologous End-Joining Mutant for Neurospora sitophila Research

Abstract Disruption of the nonhomologous end-joining (NHEJ) pathway has been shown to increase the efficiency of transgene integration into targeted genomic locations of Neurospora crassa and other fungi. Here, we report that a similar phenomenon occurs in a second Neurospora species: N. sitophila. Specifically, we show that deletion of N. sitophila mus-51 increases the efficiency of targeted-transgene integration, presumably by disrupting NHEJ. Researchers interested in obtaining the N. sitophila mus-51Δ strains described in this study can obtain them from the Fungal Genetics Stock Center (FGSC).

Spatially restricted long-term transgene expression in the developing skin used for studying the interaction of epidermal development and sensory innervation.

Skin development is tightly temporally coordinated with its sensory innervation, which consists of the peripheral branches of the dorsal root ganglion (DRG) axons. Various studies suggest that the skin produces a long-range attractant for the sensory axons. However, the exact identity of the guidance cue(s) remains unclear. To reveal the detailed molecular mechanism that controls DRG axon guidance and targeting, manipulation of specific skin layers at specific time points are required. To test a variety of attractants that can be expressed in specific skin layers at specific timepoints, we combined in utero electroporation with the Tol2 transposon system to induce long-term transgene expression in the developing mouse skin, including in the highly proliferative epidermal stem cells (basal layer) and their descendants (spinous and granular layer cells). The plasmid solution was injected as close to the hindpaw plantar surface as possible. Immediately, electric pulses were passed through the embryo to transduce the plasmid DNA into hindpaw skin cells. Balancing outcome measurements including: embryo survival, transfection efficiency, and the efficiency of transgene integration into host cells, we found that IUE was best performed on E13.5, and using an electroporation voltage of 34V. After immunostaining embryonic and early postnatal skin tissue sections for keratinocyte and sensory axon markers, we observe the growth of axons into skin epidermal layers including areas expressing EGFP. Therefore, this method is useful for studying the interaction between axon growth and epidermal cell division/differentiation.

Cre-Mediated Transgene Integration in Chinese Hamster Ovary Cells Using Minicircle DNA Vectors.

Bacterial backbone sequences of conventional plasmid vectors have been reported to exhibit negative effects on transgene expression in mammalian cells, such as cytotoxicity and gene silencing. Minicircle DNA vectors can be employed to overcome these issues and to improve the transfection efficiency because of their smaller size. In this study, transgenes are integrated into the hypoxanthine phosphoribosyltransferase (hprt) locus of Chinese hamster ovary (CHO) cells by the Cre-loxP system using minicircle DNA vectors as transgene donors. The targeted transgene integration efficiency is improved 2-3-fold (≈1.4%) using minicircle DNA vectors compared with conventional plasmid vectors. Moreover, clones with expected structures after transgene integration are obtained with a high frequency. When a transgene together with bacterial sequences derived from a plasmid vector is integrated into the hprt locus, the cell growth rate and antibody titer decrease. These results indicate that minicircle DNA vectors are more suitable than conventional plasmid vectors for transgene delivery in recombinant protein production using CHO cells.

CtIP fusion to Cas9 enhances transgene integration by homology-dependent repair

In genome editing with CRISPR–Cas9, transgene integration often remains challenging. Here, we present an approach for increasing the efficiency of transgene integration by homology-dependent repair (HDR). CtIP, a key protein in early steps of homologous recombination, is fused to Cas9 and stimulates transgene integration by HDR at the human AAVS1 safe harbor locus. A minimal N-terminal fragment of CtIP, designated HE for HDR enhancer, is sufficient to stimulate HDR and this depends on CDK phosphorylation sites and the multimerization domain essential for CtIP activity in homologous recombination. HDR stimulation by Cas9–HE, however, depends on the guide RNA used, a limitation that may be overcome by testing multiple guides to the locus of interest. The Cas9–HE fusion is simple to use and allows obtaining twofold or more efficient transgene integration than that with Cas9 in several experimental systems, including human cell lines, iPS cells, and rat zygotes.

Suppressing mosaicism by Au nanowire injector-driven direct delivery of plasmids into mouse embryos.

Transgenic animals have become key tools in a variety of biomedical research areas. However, microinjection commonly used for producing transgenic animals has several challenges such as physical and chemical damage to the embryos due to microinjector with buffer, and low transgene integration rates with frequent mosaicism. Here, we report direct delivery of plasmids into mouse embryos using a Au nanowire injector (NWI) that significantly improved transgene integration efficiency and suppressed mosaicism. The Au NWI could deliver plasmid into the pronucleus (PN) of a mouse zygote without buffer and rapidly release it with electric pulse. Because zygote, which is a fertilized 1-cell stage embryo, has two physical barriers (cytoplasmic membrane and zona pellucida), direct delivery of plasmids into PN of zygote is more difficult than into a normal cell type. To penetrate the two physical barriers with minimal disruption of the embryo, we optimized the diameter and length of Au NWI. The mosaicism is more reduced in the Au NWI injected embryos than in micropipette injected embryos, which was determined by the expression of transgene in a blastocyst stage. We suggest that Au NWI can increase the efficiency of gene delivery into zygote with suppressed mosaicism and become a useful alternative.

357 EARLY FETAL DEVELOPMENT OF NUCLEAR TRANSFER BOVINE EMBRYOS GENERATED FROM FIBROBLASTS GENETICALLY MODIFIED BY piggyBac TRANSPOSITION

Transposon-mediated transgenesis is a well-established tool for genome manipulation in small animal models. However, translation of this active transgenesis method to the large animal setting requires further investigation. We have previously demonstrated that a helper-independent piggyBac (PB) transposon system can efficiently transpose transgenes into the bovine genome [Alessio et al. 2014 Reprod. Domest. Anim. 49 (Suppl. 1), 8]. The aims of the current study were a) to investigate the effectiveness of a hyperactive version of the PB transposase, and b) to determine the ability of the genetically modified cells to support early embryo and fetal development upon somatic cell nuclear transfer (SCNT). Bovine fetal fibroblasts (BFF) were chemically transfected with either pmGENIE-3 (a helper-independent PB transposon conferring genes for hygromycin resistance and enhanced green fluorescent protein (EGFP); Urschitz et al. 2010 PNAS USA 107, 8117–8122), pmhyGENIE-3 (carrying an hyperactive version of the PB transposase; Marh et al. 2012 PNAS USA 109, 19 184–19 189), or pmGENIE-3/Δ PB (a control plasmid lacking a functional PB transposase). Upon transfection, cell cultures were subjected to 14 days of hygromycin selection. Antibiotic-resistant and EGFP+ colonies were counted and data analysed by ANOVA and Tukey's test. For SCNT, pmhyGENIE-3 and pmGENIE-3 polyclonal cell lines were selected by FACS and individual cells used as nuclear donors. Day 7 blastocysts were nonsurgically transferred to synchronized recipients. Conceptuses were recovered by Day 35 of gestation, observed under fluorescence excitation, and genotyped. The mean number of colonies in pmhyGENIE-3 group was significantly higher than those in pmGENIE-3 and the control group (324.0 ± 17.8 v. 100.0 ± 16.1 and 2.8 ± 0.8 respectively, n = 4–7; P < 0.05). The hyperactive transposase increased transgene integration efficiency 3.24 times compared with the conventional PB transposase. The SCNT and early fetal development data are summarised in Table 1. Phenotypic analysis revealed that both transgenic fetuses and the extraembryonic membranes expressed EGFP with no macroscopic evidence of variegated transgene expression. Molecular analysis by PCR confirmed that both fetuses carried the transposon DNA. Here, we demonstrate that a hyperactive version of the PB transposase is more active in bovine cells than the conventional PB transposase. In addition, SCNT embryos generated from genetically modified cells by the pGENIE transposon system can progress to early stages of fetal development. Table 1.SCNT and early fetal development of bovine fibroblasts transposed with piggyBac1 The financial support of UNRC, CONICET and ANPCyT from Argentina is gratefully acknowledged.

Characterization of DNA repair deficient strains of Chlamydomonas reinhardtii generated by insertional mutagenesis.

While the mechanisms governing DNA damage response and repair are fundamentally conserved, cross-kingdom comparisons indicate that they differ in many aspects due to differences in life-styles and developmental strategies. In photosynthetic organisms these differences have not been fully explored because gene-discovery approaches are mainly based on homology searches with known DDR/DNA repair proteins. Here we performed a forward genetic screen in the green algae Chlamydomonas reinhardtii to identify genes deficient in DDR/DNA repair. We isolated five insertional mutants that were sensitive to various genotoxic insults and two of them exhibited altered efficiency of transgene integration. To identify genomic regions disrupted in these mutants, we established a novel adaptor-ligation strategy for the efficient recovery of the insertion flanking sites. Four mutants harbored deletions that involved known DNA repair factors, DNA Pol zeta, DNA Pol theta, SAE2/COM1, and two neighbouring genes encoding ERCC1 and RAD17. Deletion in the last mutant spanned two Chlamydomonas-specific genes with unknown function, demonstrating the utility of this approach for discovering novel factors involved in genome maintenance.

TALE nickase mediates high efficient targeted transgene integration at the human multi-copy ribosomal DNA locus

Although targeted gene addition could be stimulated strikingly by a DNA double strand break (DSB) created by either zinc finger nucleases (ZFNs) or TALE nucleases (TALENs), the DSBs are really mutagenic and toxic to human cells. As a compromised solution, DNA single-strand break (SSB) or nick has been reported to mediate high efficient gene addition but with marked reduction of random mutagenesis. We previously demonstrated effective targeted gene addition at the human multicopy ribosomal DNA (rDNA) locus, a genomic safe harbor for the transgene with therapeutic potential. To improve the transgene integration efficiency by using TALENs while lowering the cytotoxicity of DSBs, we created both TALENs and TALE nickases (TALENickases) targeting this multicopy locus. A targeting vector which could integrate a GFP cassette at the rDNA locus was constructed and co-transfected with TALENs or TALENickases. Although the fraction of GFP positive cells using TALENs was greater than that using TALENickases during the first few days after transfection, it reduced to a level less than that using TALENickases after continuous culture. Our findings showed that the TALENickases were more effective than their TALEN counterparts at the multi-copy rDNA locus, though earlier studies using ZFNs and ZFNickases targeting the single-copy loci showed the reverse. Besides, TALENickases mediated the targeted integration of a 5.4kb fragment at a frequency of up to 0.62% in HT1080 cells after drug selection, suggesting their potential application in targeted gene modification not being limited at the rDNA locus.

Hyperactive self-inactivating piggyBac for transposase-enhanced pronuclear microinjection transgenesis

We have developed a unique method for mouse transgenesis. The transposase-enhanced pronuclear microinjection (PNI) technique described herein uses the hyperactive piggyBac transposase to insert a large transgene into the mouse genome. This procedure increased transgene integration efficiency by fivefold compared with conventional PNI or intracytoplasmic sperm injection-mediated transgenesis. Our data indicate that the transposase-enhanced PNI technique additionally requires fewer embryos to be microinjected than traditional methods to obtain transgenic animals. This transposase-mediated approach is also very efficient for single-cell embryo cytoplasmic injections, offering an easy-to-implement transgenesis method to the scientific community.

Optimising parameters for biolistic gun-mediated genetic transformation of tea [Camellia sinensis (L.) O. Kuntze

SummaryGenetic improvement of tea through breeding is difficult. Therefore, transgenic tea plants expressing the osmotin gene from Nicotiana tabacum were produced using parameters optimised for biolistic-gun mediated transformation. During optimisation, a total of 4,500 somatic embryos were bombarded using nine combinations of variable target distances and burst pressures, while keeping the gap distance (0.6 cm) and macrocarrier flight distance (16 mm) constant.A total of 90 independent, PCR-positive lines were generated. Southern hybridisation confirmed integration of the osmotin gene in 26 out of 27 PCR-positive lines (three independent lines from each of the nine parameter combinations were selected at random). Statistical analysis revealed that the efficiency of transgene integration was significantly affected by target distance. Only those lines derived from somatic embryos bombarded with 1.0 µg plasmid DNA using a 7.58 MPa burst pressure and 9-cm target distance showed osmotin expression. This was evident from strong northern hybridisation and RT-PCR signals. Leaves of 4-year-old transgenic plants growing in a contained polythene tunnel showed improved osmotic adjustment in response to osmotic stress imposed by NaCl. The osmotic potentials of transgenic leaves immersed in 100 mM or 200 mM NaCl solutions were more negative than those of non-transformed control leaves.

CHO cell lines generated by PiggyBac transposition

A major bottleneck in the manufacture of recombinant therapeutic proteins is the time and effort needed for the generation of stable, high-producing mammalian cell lines. Conventional gene transfer methods for stable cell line generation rely on random transgene integration, resulting in unpredictable and highly variable levels of expression of the transgene in individual clones [1,2]. As a consequence, a large number of stably transfected cells must be analyzed to recover a few high-producing clones. Recently, we described the use of a PiggyBac (PB) transposon for the generation of high-producing mammalian cell lines [3]. The PB dual vector system consists of 1) a donor vector carrying an artificial transposon with a mammalian expression cassette for the recombinant transgene and puromycin selection marker and 2) a helper vector driving transient expression of the PB transposase (PBase). PB transposition mediates stable transgene integration via a “cut and paste” mechanism in which the PBase excises the artificial transposon sequence from the plasmid and catalyzes its insertion into the host cell genome. A main advantages of the PB system over conventional passive integration are an improved efficiency of transgene integration resulting in a more integration events and more stable clones. Furthermore, PB favors transgene integration into actively transcribed regions of the host genome [4]. The PB transposon has a high cargo capacity of up to 14 Kb and transposition results in the stable genomic integration of well-defined sequences, thus reducing the probability of integration of truncated, non-functional transgenes [5]. Finally, recent reports have demonstrated the feasibility of using the PB system to obtain persistent expression of multiple genes carried either on a single or on distinct donor vectors [6]. We initially determined the efficiency of the PB system in the generation of stable lines using suspension adapted mammalian cells. CHO and HEK 293 cells were co-transfected with an eGFP-bearing donor plasmid along with the PB helper plasmid. The cells were then grown in the absence of puromycin, and the percentage of GFP-expressing cells in each culture was determined by flow cytometry on a daily basis. By 21 days post-transfection, the remaining GFP-positive cells were assumed to be recombinant. Compared to conventional transfection of plasmid DNA, PB transposition resulted in an improvement in the efficiency of stable cell line generation up to 20-fold for both CHO and HEK 293 cells (Figure ​(Figure1A1A). Figure 1 A) Enhanced stable integration efficiency in CHO and HEK 293 cells by PB transposition. B) Productivity analysis of CHO clonal cell lines sorted from cell pools generated by PB transposition (PB) or conventional transfection (TX). C) Analysis of the stability ... To further evaluate the PB system, CHO cells expressing a tumour necrosis factor receptor:Fc fusion protein (TNFR:Fc) were generated either by PB-transposition or by conventional transfection. Clonal cell lines were recovered following selection in 50 or 10 µg/mL puromycin for two weeks. Recovered lines were grown in suspension culture for 7 days in 24-well plates after which the medium was analyzed by ELISA to determine TNFR:Fc productivity. Transposition increased the frequency of high-producing clones in the transfected population (Figure ​(Figure1B).1B). To further characterized for the level and stability of transgene expression the original cell pools generated by PB transposition or conventional transfection, as well as the top 4 producers from each transfection were cultivated in the absence of selection in serum-free suspension culture, over a period of 16 or 14 weeks, respectively. When compared to clones and cell pools generated by conventional transfection, PB-derived cell lines and cell pools produced up to 4-fold more recombinant protein and had greater transgene expression stability (Figure ​(Figure1C1C) In conclusion our results demonstrate that stable cell lines derived by PB transposition are efficiently generated and are more productive than cell lines generated by conventional transfection methods. Therefore, the PB system represents a valuable and practical alternative to standard plasmid transfection to efficiently generate cell clones with stable and enhanced transgene expression.

Comparative Genomic Integration Profiling of Sleeping Beauty Transposons Mobilized With High Efficacy From Integrase-defective Lentiviral Vectors in Primary Human Cells

It has been previously shown that integrase-defective HIV-1-based gene vectors can serve, with moderate efficiency, as substrate for DNA transposition by a transiently expressed Sleeping Beauty (SB) transposase. Here, we describe the enhanced gene transfer properties of a HIV-1/SB hybrid vector that allows efficient DNA transposition, facilitated by the hyperactive SB100X transposase, from vector DNA intermediates in primary human cells. Potent transposase-dependent integration of genetic cargo carried by the hybrid HIV-1/SB vector (up to 160-fold above background) is reported in human cell lines as well as in primary human fibroblasts and keratinocytes. The efficiency of transgene integration in context of the newly developed hybrid vector is comparable with that of conventional lentiviral vectors (LVs). Integration profiles of integrating HIV-1-derived vectors and SB transposons mobilized from LVs are investigated by deep sequencing of a large number of integration sites. A significant bias of lentiviral integrations in genes is reported, confirming that biological properties of the viral integration machinery facilitate preferred insertion into actively transcribed genomic regions. In sharp contrast, lentiviral insertions catalyzed by the SB100X transposase are far more random with respect to genes. Based on these properties, HIV-1/SB vectors may become valuable tools for genetic engineering and therapeutic gene transfer.

Quality of transgenic rabbit embryos with different EGFP gene constructs

The aim of this study was to compare the quality of rabbit transgenic embryos obtained upon microinjection of gene constructs containing different promoters and green fluorescent proteins (CMVIE-EGFP, PGK-EGFP and CMVIE-hrGFP). Developmental rate, total cell number in hatching blastocyst stage, number of apoptotic cells, diameter of embryos, transgene integration and transgenic mosaicism were investigated.The rate of rabbit embryos microinjected with the different gene constructs developed up to morula stage was significantly lower (p < 0.05) than that of intact (non-microinjected) rabbit embryos (66-74vs. 98%). The highest efficiency of transgene integration (15%) was found when the CMVIE-EGFP (DrdI) gene construct was used, however a significantly higher transgenic mosaicism (60%) was found in rabbit embryos using this gene. The lowest cell number was counted in rabbit transgenic embryos with CMVIE-rhGFP linearized by ScaI (115.0 ± 8.20), the highest cell number (134.0 ± 35.00) was detected in rabbit transgenic embryos carrying PGK-EGFP (Not I) gene. The highest number of apoptotic cells (2.6 ± 0.33) was recorded in rabbit transgenic embryos with the integrated CMVIE-EGFP (ClaI) transgene.Based on these results a more suitable gene marker for rabbit transgenic embryos production and selection is the CMVIE-EGFP (ClaI) gene construct. Prior to using microinjected embryos (for embryo transfer, vitrification or ESC isolation) it is necessary to pre-select microinjected embryos with evident transgenic mosaicism.

High frequency Agrobacterium tumefaciens-mediated plant transformation induced by ammonium nitrate

Success in plant genetic transformation depends on the efficiency of explant regeneration and transgene integration. Whereas the former one depends on explant totipotency, the latter depends on the activity of host DNA repair and chromatin organisation factors. We analyzed whether factors that result in an increase in recombination frequency can also increase transformation efficiency. Here, we report that a threefold increase in the concentration of NH(4)NO(3) in the growth medium results in more than a threefold increase in the Agrobacterium tumefaciens-mediated transformation frequency of Nicotiana tabacum plants. Regeneration of calli without selection showed that the increase in transformation frequency was primarily due to the increase in transgene integration efficiency rather than in tissue regeneration efficiency. PCR analysis of insertion sites showed a decrease in the frequency of truncations of the T-DNA right border and an increase on the left border. We hypothesize that exposure to ammonium nitrate modifies the activity of host factors leading to higher frequency of transgene integrations and possibly to the shift in the mechanism of transgene integrations.

In vivoGene Transfer into Testis and Sperm: Developments and Future Application

Despite significant advances in the treatment of infertility via assisted reproductive technology (ART), the underlying causes of idiopathic male infertility still remain unclear. Accumulating evidence suggests that disorders associated with testicular gene expression may play an important role in male infertility. To be able to fully study the molecular mechanisms underlying spermatogenesis and fertilization, it is necessary to manipulate gene expression in male germ cells. Since there is still no reliable method of recapitulating spermatogenesis culture, the development of alternative transgenic approaches is paramount in the study of gene function in testis and sperm. Established methods of creating transgenic animals rely heavily upon injection of DNA into the pronucleus or the injection of transfected embryonic stem cells into blastocysts to form chimeras. Despite the success of these two approaches for making transgenic and knockout animals, concerns remain over costs and the efficiency of transgene integration. Consequently, efforts are in hand to evaluate alternative methodologies. At present, there is much interest in developing approaches that utilize spermatozoa as vectors for gene transfer. These approaches, including testis mediated gene transfer (TMGT) and sperm mediated gene transfer (SMGT), have great potential as tools for infertility research and in the creation of transgenic animals. The aim of this short review is to briefly describe developments in this field and discuss how these gene transfer methods might be used effectively in future research and clinical arenas.

Methods to produce marker‐free transgenic plants

Selectable marker genes (SMGs) have been extraordinarily useful in enabling plant transformation because of the low efficiency of transgene integration. The most used SMGs encode proteins resistant to antibiotics or herbicides and use negative selection, i.e., by killing nontransgenic tissue. However, there are perceived risks in wide-scale deployment of SMG-transgenic plants, and therefore research has recently been performed to develop marker-free systems. In this review, transformation using markers not based on antibiotic or herbicide resistance genes, as well as different systems of marker gene deletion, are discussed.

The effect of coating single- and double-stranded DNA with the recombinase A protein of Escherichia coli on transgene integration in mice

Embryo survival and transgene integration rates are two major factors that influence the efficiency of transgenic animal production by pronuclear microinjection. Recombinase A protein-coated transgenes were compared for transgene integration and embryo survival with their non-coated counterparts in both single- and double-stranded forms. Murine zygotes were microinjected with a large 30 kb alpha(S1)-casein/human lysozyme DNA construct and a small 5.5 kb beta-lactoglobulin/desaturase DNA construct using four different construct preparations for each gene. The preparations included recombinase A protein-coated, single- and double-stranded DNA constructs and non-coated, single- and double-stranded DNA constructs. Using conventional non-coated, double-stranded DNA constructs, we obtained a transgene integration efficiency of 1.5% (1352 embryos transferred produced 20 transgenic pups). The same double-stranded DNA constructs coated with recombinase A protein yielded a similar percentage of transgene integration (1.1%, 18/1697). Using single-stranded DNA, non-coated constructs produced a transgene integration rate of 0.5%, while none of the 1040 zygotes injected with recombinase A-coated constructs produced transgenic pups. While recombinase A protein coating produced no effect on embryo survival, litter size or pregnancy rate with double-stranded constructs, a detrimental effect was observed on embryo survival (P < 0.001) and pregnancy rate (P < 0.005) with recombinase A protein coating of single-stranded human lysozyme DNA constructs. A trend toward increased embryo survival (P = 0.054) with no difference in pregnancy rate (P > 0.05) was observed with the recombinase A protein coating of single-stranded desaturase constructs. These results suggest that recombinase A protein coating of single- and double-stranded DNA constructs produced no significant differences (P > 0.05) in the efficiency of generating transgenic mice with respect to the percentage of transgenic animals born.

410 USE OF SPERMATOZOA AS VECTORS OF EXOGENOUS DNA FOR IN VITRO PRODUCTION OF BOVINE TRANSGENIC EMBRYOS

There are many methods to produce transgenic animals, although when considering bovine species, those methods offer low repeatability, high costs, and low transgene integration efficiency. To overcome these difficulties, sperm mediated gene transfer (SMGT) could be used as an alternative to produce transgenic embryos. This technology allows carrying exogenous DNA into the oocyte during the fertilization period, once spontaneous binding exists between spermatozoa and exogenous DNA. The aim of this study was to compare 4 methods for incorporating DNA into sperm cells—sperm incubation, capacitation, electroporation, and lipofection—to verify the efficiency of embryo production with exogenous DNA, employing spermatozoa as a vector. Cumulus–oocyte complexes (COCs) from abattoir-derived bovine ovaries were randomly divided into 5 groups (4 experimental groups: sperm incubation, sperm capacitation with calcium ionophore, electroporation, and lipofection; and 1 control group). In vitro maturation was performed in TCM-199 medium supplemented with 10% FCS, sodium pyruvate, gentamycin, FSH, hCG, and estradiol in an incubator at 39�C, in an atmosphere of 5% CO2 in air and high humidity for 24 h. After Percoll gradient (45/90%) separation at 600g for 30 min, spermatozoa were washed in Talp Semen medium (200g for 5 min). For in vitro fertilization (IVF), 5 � 106 spermatozoa were used to inseminate microdroplets with 20 matured oocytes from all groups: incubation with exogenous DNA for 1 h, sperm capacitation (250 nM of calcium ionophore) for 5 min followed by sperm incubation for 1 h, electroporation (500V), and lipofection (Effectene�; Qiagen, Mississauga, Ontario, Canada). The EYFP-Nuc plasmid (500 ng mL-1; Clontech, Mountain View, CA, USA) was used as exogenous DNA. Oocytes inseminated with non-treated sperm were considered as the control group. The presumptive zygotes were co-cultured with a granulosa cell monolayer in SOFaa medium in an incubator at 39�C, with an atmosphere of 5% CO2 in air and high humidity. The blastocyst rate was analyzed by ANOVA. Embryos from all experimental and control groups were subjected to PCR to detect internalized EYFP, using primers specific to this exogenous DNA, and considering positive embryos those that showed a fragment of 440 bp after electrophoresis. The fragment of 440 bp from positive embryos was sequenced to check correspondence to the EYFP. Electroporation (17.95%) and sperm capacitation (15.12%) were more efficient for EYFP-positive embryo production when compared to the lipofection protocol (6.25%). Sperm incubation (12.5%) did not show a significant difference from the other groups. Sequenced PCR-positive products for EYFP showed 100% homology with nucleotides of EYFP at GenBank. In conclusion, it is possible to use SMGT to deliver exogenous DNA into an oocyte at the time of IVF. This work was supported financially FAPESP 03/08542-5 and 03/07456-8.

Strain Differences in Superovulatory Response, Embryo Development and Efficiency of Transgenic Rat Production

The differences between rat strains in superovulation response, in vitro and in vivo development of preimplantation embryos and overall transgenic efficiency was studied. The protocols for induction of superovulation using single injections of pregnant mare's serum gonadotropin (PMSG) or minipumps with follicle stimulating hormone (FSH) were compared in Lewis (LEW), Wistar-Kyoto (WKY), and stroke-prone spontaneously hypertensive rats (SHRSP) or Sprague-Dawley (SD) and Wistar rats as representative inbred or outbred strains, respectively. The percentage of mated animals with positive superovulatory response was similar in all strains (60.0-100%). The mean number of ova per donor was not dependent on the kind of hormonal treatment used within each rat strain. In general, females from outbred SD and Wistar rats were more responsive to hormonal treatments than animals from inbred rat strains. In addition, SD female rats produced a significantly higher number of embryos per female in response to PMSG-treatment compared to all other strains. Between the inbred strains, SHRSP was the most effective for superovulation. In vitro development of intact zygotes to the blastocyst stage was not different between SD, Wistar and SHRSP rats. In contrast, in vitro development of WKY zygotes was significantly less efficient than in other strains. However, 2-cell stage embryos in vivo produced from SD, SD x Wistar and WKY animals showed no difference in competence to develop to blastocyst stage in vitro. The proportion of offspring developing after oviduct transfer of intact zygotes was similar in all strains (44.0-56.4%) with the exception of WKY rats (35.9%). We also compared the survival rate after injection, ability of manipulated zygotes to develop to term and overall transgenic efficiency in various rat strains. SD and SHRSP zygotes survived after microinjection better than the WKY and Lewis zygotes. No differences were found in the efficiency of transgene integration per newborn in different strains ranging from 5.7 to 16.7%. The results of this study demonstrate that different rat strains have varying responses to superovulation, sensitivity to microinjection, capability to develop in vitro until blastocyst stage or in vivo to term after transfer to foster mothers. Despite these differences all studied strains can be used for efficient transgenic rat production.

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 microg/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 microg/mL rhFVIII in milk, with an activity of 0.594 IU/mL. The DM founder female produced 118 microg/mL rhFVIII, with activity values of 18 IU/ 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.