19 DIRECT INTRODUCTION OF GENE CONSTRUCTS INTO THE PRONUCLEUS-LIKE STRUCTURE OF CLONED EMBRYOS: A NEW STRATEGY FOR THE GENERATION OF GENETICALLY MODIFIED PIGS

Because of a rising demand for complex porcine disease models for biomedical research, the approaches for their generation need to be adapted. In this study we describe the direct introduction of a gene construct into the pronucleus (PN)-like structure of cloned embryos as a new strategy for the generation of genetically modified pigs, termed “nuclear injection.” This new strategy could allow adding large constructs into cloned embryos with a genetically modified background. Moreover, the generation of multiple transgenic pigs based on already existing transgenic cells could be facilitated due to a reduction of recloning steps. To evaluate the reliability of this approach, developmental ability of the embryos in vitro or in vivo and integration or expression efficiency of the transgene were examined. Somatic cell NT using in vitro matured oocytes was performed. Wild-type cells were used as nuclear donors. Centrifugation was done 10 h after activation for visualisation of a PN-like structure. Subsequently, linearized pmaxGFP (10 ng μL–1; Amaxa Biosystems) was directly injected into the PN-like structure of the cloned embryos. Expression efficiency in blastocysts generated by nuclear injection was compared to blastocysts generated by the classical PN injection using in vitro-produced zygotes. Injected embryos were transferred to recipient pigs without green fluorescent protein (GFP) selection, and fetuses collected at Day 68 were characterised for their integration and expression pattern of the transgene. Eighty percent of the reconstructed embryos (633/787) exhibited a PN-like structure, which made them available for the method. Green fluorescent protein fluorescence was observed in about half of total blastocysts (52.5%, 21/40), which was comparable to classical PN injection (68.4%, 28/41). Green fluorescent protein fluorescence of blastocysts ranged from mosaic to uniform patterns. In total, 478 pmaxGFP-injected embryos were transferred into 4 recipients, 4 fetuses were collected from one of them. In one of the fetuses that developed normally, the integration of the transgene was confirmed by PCR in different major organs from all 3 primary germ layers and placenta. The integration pattern of the transgene was mosaic (43 out of 84 single-cell colonies established from kidney were positive for GFP DNA by PCR). However, the proportion of GFP-expressing cells was very low (5 out of 84 colonies expressed GFP), which might indicate silencing of transgene expression. Our pilot study demonstrated that the direct introduction of gene constructs into cloned embryos could be a new strategy for the generation of genetically modified pigs. This approach could also be applied to rescue embryos with lethal knockouts by transfer of corresponding human genes, to generate pigs as bioreactors, e.g. for antibodies. This work was supported by the German Research Council – Transregio Collaborative Research Center 127 “Xenotransplantation.”

40 EARLY DEVELOPMENT OF RECONSTRUCTED MOUSE EMBRYOS USING BONE MARROW CELLS FROZEN WITHOUT CRYOPROTECTANT

Animal cells frozen with suitable cryoprotectants have been successfully cryopreserved for long periods of time, maintaining viability upon thawing. Animal cells frozen without cryoprotectant, however, may suffer serious damage and not be useful as donors in somatic cell nuclear transfer (SCNT). However, in some cases, old animal samples were frozen only as a whole body or a piece of tissue without cryoprotectant. If the cells from such old samples could be useful for SCNT, then there are potentially many candidates where individual animals could be reproduced. In this study, we examined the possibility of using mouse bone marrow cells frozen without cryoprotectant as nuclear donors in SCNT. Thigh bones were collected from B6C3F1 mice and frozen in either a –25�C or a –80�C freezer for more than one month. Thawing of frozen bones was performed by placing them in an incubator at 37�C. Bone marrow cells were collected by washing the bone cavity with saline. Recipient oocytes for SCNT were collected from B6D2F1 female mice. The enucleation of recipient oocytes and the injection of nuclei were performed as previously reported (Wakayama et al. 1998 Nature 394, 369–374) with a piezo-actuated micromanipulator system. In this study, 4 groups of mouse cells (fresh bone marrow cells, bone marrow cells frozen at –25�C, bone marrow cells frozen at –80�C, and fresh cumulus cells) were used as the nuclear donors in SCNT. After nuclear injection, embryos were kept in mCZB medium for 1 h at 37�C. Subsequently, embryos were cultured for 3 h with 5 µg mL–1 cytochalasin B and 10 mm SrCl2 for activation and cultured for an additional 20 h in mKSOM medium. The nuclear dynamics of SCNT embryos in each donor cell group was observed using 42,6-diamidino-2-phenylindole (DAPI) staining and a fluorescent microscope at 0, 1, 7, and 24 h after nuclear injection. Data were analyzed by Student's t-test. The cell viability after thawing by trypan blue vital staining was about 20% regardless of freezing temperature. At 7 h after nuclear injection, the SCNT embryos injected with frozen bone marrow cells, regardless of freezing temperature, had more single pronuclei (67%, 54/81; P < 0.05) than SCNT embryos injected with either fresh bone marrow cells (36%, 26/73) or cumulus cells (28%, 67/236). At 24 h after nuclear injection, fewer SCNT embryos injected with bone marrow cells, either fresh or frozen, developed to the 2-cell stage (fresh: 11%, 6/56; frozen at –25�C: 21%, 5/24; frozen at –80�C: 20%, 10/49) than SCNT embryos injected with cumulus cells (58%, 185/319; P < 0.05). There was no difference in the embryonic development to the 2-cell stage among SCNT embryos injected with either fresh or frozen bone marrow cells. Further studies are required to determine whether cells frozen without cryoprotectant are capable of resulting in viable clones.

Nuovi problemi derivanti dall’impiego delle tecniche di fecondazione artificiale

Le tecniche di fecondazione artificiale (PMA) pongono al medico che opera nel campo delle metodiche della riproduzione e al bioeticista notevoli problemi di ordine morale, per la cui risoluzione è necessaria una base scientifica a monte della valutazione etica. Questo studio analizza, dunque, gli aspetti più propriamente scientifici in merito alla Intra Cytoplasmic Sperm Injection (ICSI). Tale tecnica è oggi quella più usata nei centri di PMA ed ha rivoluzionato l’approccio alla sterilità di coppia. Pur tuttavia essa pone notevoli problemi, sia in merito all’impiego di seme di soggetti infertili sia connessi alla stessa tecnica. Per quanto riguarda i primi, si distinguono aspetti correlati: 1. alla presenza di anomalie citogenetiche parentali e delle cellule spermatiche, onde è stato dimostrato che pazienti infertili ammessi a programmi ICSI hanno un alto tasso di aneuploidie negli spermatozoi, inversamente correlate con i parametri seminali e il numero di forme morfologicamente normali dopo selezione; 2. all’esistenza di microdelezioni del cromosoma Y. Su tale aspetto ci si sofferma, evidenziando il ruolo dei segmenti genici coinvolti, nonché il rischio di trasmissione della microdelezione ai figli concepiti via ICSI. Sono quindi descritte le principali problematiche connesse sia alla tecnica sia all’abilità dell’operatore, evidenziando il carattere di sperimentalità della tecnica in esame. Considerando, poi, i risultati delle procedure ICSI, occorre considerare sia il tasso di fecondazione e l’outcome ostetrico sia il seppur lieve aumento di incidenza di malformazioni ed anomalie citogenetiche. Inoltre, il tentativo di ottenere gravidanze da situazioni maschili sempre più severe, pone il problema di valutare i risultati in rapporto al quadro eziologico di partenza, ma vi sono poche osservazioni in tal senso. In conclusione vengono valutati i nuovi campi di indagine, con particolare riferimento alla round spermatid injection (ROSI), alla round spermatid nuclear injection (ROSNI) ed all’impiego di spermatogoni, fino alle forme di “semi-clonazione”, e gli ulteriori problemi che permangono ancora aperti, quali gli aspetti immunologici o la trasmissibilità dell’infezione da Human Immunodeficency Virus (HIV). ---------- The reproductive technologies (RTs) puts to the physician, involved in the reproductive medicine, and the bioethicist remarkable moral problems; therefore a solid scientific ground is necessary for an ethical evaluation. This study analyses the more properly scientific aspects, particularly related to Intracytoplasmic Sperm Injection (ICSI). Such technique, that is today the more used in the RTs centres, upsets the approach to couple sterility. It places, however, remarkable problems, both about the use of infertile man sperm and about the same technique. As to the first point, we can distinguish aspects connected to the presence of parental citogenetic anomalies and in sperm cells, so it was demonstrated that infertile patients admitted to ICSI programs have a high rate of aneuploidies in the spermatozoa, inversely correlated with the sperm parameters and the number of morphologically normal shapes after selection, and to the existence of micro-deletion of the Y chromosome. About this we meant to stop to us, evidencing the role of the deleted genic segments and the risk of transmission of the microdeletion to sons conceived via ICSI. Then the authors describe the major problems connected to the technique and to the ability of the operator, evidencing the experimentation of this technology. Considering, then, the outcomes of ICSI procedures, it is necessary to consider both the fecondation rate and the obstetrical outcome, and the slight increase of the incidence of malformations and citogenetical anomalies. Moreover, the attempt to obtain pregnancies from more and more severe male situations places the problem to estimate the outcome referred with the etiology, but there are only few observations about it. In conclusion we considered the new perspectives, specially about round spermatid injection (ROSI), round spermatid nuclear injection (ROSNI) and the use of spermatogonia, until “the semi-cloning reproduction” and the other problems still opened, as the immunological aspects or the transmission of the Human Immunodeficiency Virus (HIV).

31 FULL-TERM AND LIVE RABBIT CLONES PRODUCED BY SOMATIC CELL NUCLEAR TRANSFER

Transgenic/knockout (KO) rabbits can serve as an excellent animal model for human cardiovascular diseases (CVD) and other diseases. However, the production of transgenic/KO rabbits is hindered by low efficiency of traditional DNA microinjection and the unavailability of embryonic stem cell lines. An alternative approach is to produce transgenic/KO rabbits by somatic cell nuclear transfer (SCNT) using genetically modified somatic cells as nuclear donors. Our initial objective of the study was to prove the feasibility of cloning rabbits by SCNT because rabbit is a difficult species to be cloned. Rabbit oocytes were flushed from the oviducts of superovulated donors treated with the regime of follicle-stimulating hormone (FSH) and human choriani gonadotropin (hCG). Cumulus cells were then denuded from the oocytes by incubation in 0.5% hyaluronidase and pipetting. Oocyte enucleation was conducted in M199 + 10% fetal bovine serum (FBS) and confirmed by fluorescence microscopy. Cumulus cells used for nuclear donors were prepared from fresh cumulus-oocytes complexes. The donor nucleus was transferred into a recipient oocyte by either cell fusion or direct nuclear injection method. In the cell fusion method, a small donor cell with the diameter approximately 15–19 µm was transferred into the perivitelline space of an enucleated oocyte; subsequently the somatic cell-cytoplast pair was fused by applying three direct current pulses at 3.2 kV/cm for a duration of 20 µs/pulse. In the direct nuclear injection method, a mechanically lysed donor cell was injected into oocyte cytoplasm with the aid of a piezo-drill system. Fused embryos or injected oocytes were activated by the same electrical stimulation regime described above, and subsequently cultured in M199 + 10% FBS containing 2.0 mM 6-dimethylaminopurine (DMAP) and 5 µg/mL cycloheximide for 2 h. For the in vitro study, cloned embryos were cultured in B2 medium plus 2.5% FBS for 5 days (initiation of activation = day 0) at 38.5°C in 5% CO2 humidified air. For the in vivo study, cloned embryos were cultured for 20–22 h in vitro before transfer into pseudopregnant rabbit recipients. Pregnancy was monitored by palpation and/or ultrasound on Days 14–16 post embryo transfer (ET). The results (Table 1) show that the donor nuclei-introducing rate was higher with nuclear direct injection than with the cell fusion method (P < 0.05). There were no significant differences among subsequent cleavage and development to morula and blastocysts between both methods, although the development rates of cloned embryos via electrically mediated fusion were higher than those derived from the injection group. One recipient in the injection group (1/6, 17%) and six recipients in the fusion group (6/16, 38%) were diagnosed as pregnant. From the fusion group, one full-term but stillborn and one live and healthy clone rabbit were delivered on Days 33 and 31 post-ET, respectively. To our knowledge, this is the second report of full term development of cloned rabbit by somatic nuclear transfer cloning. Our further study is to clone live rabbit offspring with modified transgenic/KO somatic cell lines. Table 1. In vitro development of rabbit cloned embryos with cumulus cells as nuclear donors This work was supported by NIH/NCRR-SBIR grant: 1R43RR020261–11.

Nup153 Affects Entry of Messenger and Ribosomal Ribonucleoproteins into the Nuclear Basket during Export

A specific messenger ribonucleoprotein (RNP) particle, Balbiani ring (BR) granules in the dipteran Chironomus tentans, can be visualized during passage through the nuclear pore complex (NPC). We have now examined the transport through the nuclear basket preceding the actual translocation through the NPC. The basket consists of eight fibrils anchored to the NPC core by nucleoprotein Nup153. On nuclear injection of anti-Nup153, the transport of BR granules is blocked. Many granules are retained on top of the nuclear basket, whereas no granules are seen in transit through NPC. Interestingly, the effect of Nup153 seems distant from the antibody-binding site at the base of the basket. We conclude that the entry into the basket is a two-step process: an mRMP first binds to the tip of the basket fibrils and only then is it transferred into the basket by a Nup153-dependent process. It is indicated that ribosomal subunits follow a similar pathway.

Chromosomal Storage of the RNA-editing Enzyme ADAR1 in Xenopus Oocytes

ADARs (adenosine deaminases that act on RNA) are RNA-editing enzymes that convert adenosines to inosines in structured or double-stranded RNAs. Expression and intracellular distribution of ADAR1 is controlled by a plethora of mechanisms suggesting that enzyme activity has to be tightly regulated. Mammalian ADAR1 is a shuttling protein, whereas Xenopus ADAR1 is exclusively nuclear. In oocytes, Xenopus ADAR1 associates with most nascent transcripts but is strongly enriched at a specific site on chromosome 3, termed the special loop. Enrichment at this site requires the presence of RNAs but is independent of ongoing transcription. Here we show that RNAs transcribed elsewhere in the genome accumulate at the special loop even in the absence of transcription. In situ hybridization experiments, however, indicate the absence of known editing substrates from this site. In the absence of transcription also other RNA binding and processing factors accumulate at the special loop, suggesting that ADAR1 is stored or assembled at the special loop in an RNA-containing complex. Nuclear injection of RNAs providing binding sites for ADAR1 dissociates the enzyme from the special loop, supporting the notion that the special loop represents a site where ADAR1 is stored, possibly for later use during development.