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

Transgenesis is a complex process. Among the most important factors affecting the production of potential transgenic pigs using standard DNA microinjection into zygote pronuclei are the type of promoter used to express the gene construct and the method of zygote transfer used for transformation. The transformation of zygotes using different gene constructs showed that the promoter type infl uenced the developmental capability of the transformed zygotes, and this affected the percentage of born piglets. The transfer of transformed zygotes into a single oviduct resulted in a higher percentage of potential transgenic piglets born, this fi gure was almost doubled compared with transfer into both oviducts. Recently, research into the production of transgenic pigs has shown that factors connected with reproduction physiology and embryological factors such as the number of zygotes transferred after transformation and the season of the year have a signifi cant infl uence but are not critical to the effi ciency of production of transgenic pigs. The best results were obtained when less than 20 transformed zygotes per recipient were transplanted and the autumn season is the time when the environmental factors are optimal for production of potential transgenic pigs.

The objective of this study was to examine whether the ICSI-mediated gene transfer method using in vitro matured oocytes and frozen sperm head could actually produce transgenic pigs. We also aimed at examining whether transgenic pigs can be cloned from somatic cells of a transgenic pig generated by the ICSI-mediated method. A bicistronic gene constituted of the human albumin (hALB) and enhanced green fluorescent protein (EGFP) genes was introduced into pig oocytes by the ICSI-mediated method. Transfer of 702 embryos produced by the ICSI-mediated method into five gilts resulted in 4 pregnancies. When three of the recipients, which had received total 312 of the embryos were autopsied, 32 including 1 transgenic fetuses were obtained. One of the recipients gave birth to three live piglets including one transgenic pig, showing a strong green fluorescence in the eyeballs, oral mucous membrane and subcutaneous tissues. Fluorescent microscopy revealed uniform GFP expression in all cell lines established from kidney, lung and muscle of the founder transgenic pig obtained. Nuclear transfer of these cells resulted in stable in vitro development of cloned embryos into the blastocyst stage, ranging from 12.9 to 19.8%. When 767 of the nuclear transfer embryos were transferred to 5 recipients, all became pregnant and gave birth to a total of six live transgenic-clones. The transgene copy number and integrity in the founder pig were maintained in the primary culture cells established from the founder as well as in the clones produced from these cells. Our study demonstrates that the ICSI-mediated gene transfer is an efficient and practical method to produce transgenic pigs, using frozen sperm heads and in vitro matured oocytes. It was also shown that combination of ICSI-mediated transgenesis and nuclear transfer is a feasible technology of great potential in transgenic pig production.

ABSTRACTThe generation of transgenic pigs is an entirely new way of breeding. In contrast to classical breeding techniques the objects of manipulation in this case are individual genes rather than the entire genome of an organism. In pigs DNA-microinjection into the pronuclei of zygotes is the only available technique of transferring genetic material developed so far. The process involves collection, manipulation, microinjection, cultivation, and transfer of early embryos and also molecular-biological techniques allowing cloning of gene constructs, preparation of suitable injection solutions, and techniques allowing detection of integrated and expressed transgenes in transgenic animals. Gene transfer in pigs usually yields less than 1% transgenic piglets per injected zygote. Our own experiments have shown that simultaneous transfer of untreated control embryos increases yields from 0.5% to 1%.Gene transfer in pigs can be employed in particular to increase growth performance and carcass composition by using genes encoding hormones of the growth hormone cascade (GHRH, GH, IGF-I). So far, the effects already known from experiments in mice have not been reproduced in pigs.We are currently investigating whether the transfer of the influenza resistance gene Mx+of mice will yield disease-resistant pigs.Breeding with transgenic animals must take into account that approximately 30% of the primary transgenic animals will be mosaics which will not pass on the transgene to their offspring. Unwanted side effects may also occur during gene transfer. Most important examples are instability of integrated transgenes and variability of gene expression over many generations.In about 5% of all primary transgenic animals integration of the transgene can be assumed to lead to the generation of insertion mutations. Animals carrying these mutations should not be used for breeding. Furthermore severe health problems may be caused by uncontrolled over-expression of the transgene.Much more work will be necessary in future before we will be able to employ gene transfer techniques in practical breeding programmes.

Somatic cloning is emerging as a new biotechnology by which the opportunities arising from the advances in molecular genetics and genome analysis can be implemented in animal breeding. Significant improvements have been made in SCNT protocols in the past years which now allow to embarking on practical applications. The main areas of application of SCNT are: Reproductive cloning, therapeutic cloning and basic research. A great application potential of SCNT based cloning is the production of genetically modified (transgenic) animals. Somatic cell nuclear transfer based transgenic animal production has significant advances over the previously employed microinjection of foreign DNA into pronuclei of zygotes. This cell based transgenesis is compatible with gene targeting and allows both, the addition of a specific gene and the deletion of an endogenous gene. Efficient transgenic animal production provides numerous opportunities for agriculture and biomedicine. Regulatory agencies around the world have agreed that food derived from cloned animals and their offspring is safe and there is no scientific basis for questioning this. Commercial application of somatic cloning within the EU is via the Novel Food regulation EC No. 258/97. Somatic cloning raises novel questions regarding the ethical and moral status of animals and their welfare which has prompted a controversial discussion in Europe which has not yet been resolved.

The production of transgenic pigs using somatic cell nuclear transfer (scNT) has been widely described, but a technique for removing nontransfected donor cells and for creating different founder animals has not yet been fully elucidated. In this study, four different expression vectors (pBC1hEPO, pMARBC1hEPO, pBC1hEPOwpre and pMARBC1hEPOwpre) were compared to determine the highest transgene expression, ideal conditions of enrichment of recombinant cells in vitro and efficiency of transgenesis following transfection into HC11 mammary epithelial cells. The highest protein expression in HC11 cells was obtained from the pMARBC1hEPOwpre expression vector. Next, we evaluated the efficiency of transgenic pig production by using geneticin (G418) selection alone or by using real-time PCR selection following G418 selection. Ideal enrichment of recombinant cells was obtained by a combination of real-time PCR and G418 selection; embryos reconstructed using donor cells selected by a combination of real-time PCR and G418 selection gave rise to nine piglets, all of which were transgenic. Among them, three founder transgenic pigs were established. Exogenous DNA fragments were shown to be integrated into chromosomes 1q2.4, 1p2.3 and 6q2.4, respectively, in these three pigs. However, the transgenic rate using G418 selection alone was only 33% (two of six pigs) and showed a very low efficacy compared with that of the combination of real-time PCR and G418 selection. Our results provide a valuable experimental model for applying and evaluating transgenic technology in pigs.

Pigs are considered the most likely source of organs for xenotransplantation due to their anatomical and physiological similarities to humans. Production of transgenic pigs including addition of human complement-regulatory protein genes and deletion of alpha-1,3-galactosyl transferase gene may overcome hyperacute rejection (HAR), the first and currently the most critical immunological hurdle in the development of xenogeneic organs for human transplantation. However, even after resolving HAR in pig-to-human xenotransplantation, a series of other transgenic pigs may be required to alleviate subsequent acute and chronic rejection and incompatibility of porcine proteins to human counterparts. The production of transgenic pigs is not only labor-intensive, time-consuming, and costly, but also the usefulness of such pigs in transplantation to humans is unpredictable. For these reasons, development of a reliable in vitro procedure to pre-evaluate effectiveness of the transgenic approach would be beneficial. This study was preformed to establish an in vitro model of xenotransplantation using porcine embryonic germ (EG) cells, undifferentiated stem cells derived from culture of primordial germ cells. Porcine EG cells were maintained in feeder-free state in DMEM containing 15% (v/v) fetal bovine serum and 1000 units/mL leukemia inhibitory factor. Human complement down-regulator hCD46 (also known as MCP, membrane cofactor protein) gene under the regulation of cytomegalovirus promoter was introduced into porcine EG cells. Transfected cells were selected by antibiotic treatment and confirmed by PCR. To test the resistance of hCD46-transgenic EG cells to human xenoreactive natural antibody and complement, EG cells were cultured for 1.5 days in DMEM containing 15% (v/v) normal human serum. The treatment with human serum did not affect the survival of hCD46-transgenic EG cells, whereas with the same treatment approximately one half of non-transfected EG cells failed to survive (P < 0.01). Transgenic EG cells presumably capable of overcoming HAR were used as nuclear donors for subsequent transfer of nuclei into enucleated oocytes. Among 110 reconstituted oocytes, 19 (17.3%) developed to the blastocyst stage. Analysis of individual nuclear transfer embryos by PCR indicated that 89.5% (17/19) of embryos contained transgene hCD46. The PCR-negative embryos might be due to an incomplete antibiotic selection of cells after transfection. Overall, the results of the present study demonstrate that the cell culture-based model of xenotransplantation may validate the usefulness of particular transgenic pigs prior to actual production. Further experiments on differentiation of transgenic EG cells into various cell types, cytolytic analysis of such cells to assess efficiency of xenotransplantation, and subsequent production and transfer of transgenic clone embryos to recipients may provide a useful new procedure to accelerate xenotransplantation research.

In the last few decades, transgenic animal technology has witnessed an increasingly wide application in animal breeding. Reproductive traits are economically important to the pig industry. It has been shown that the bone morphogenetic protein receptor type IB (BMPR1B) A746G polymorphism is responsible for the fertility in sheep. However, this causal mutation exits exclusively in sheep and goat. In this study, we attempted to create transgenic pigs by introducing this mutation with the aim to improve reproductive traits in pigs. We successfully constructed a vector containing porcine BMPR1B coding sequence (CDS) with the mutant G allele of A746G mutation. In total, we obtained 24 cloned male piglets using handmade cloning (HMC) technique, and 12 individuals survived till maturation. A set of polymerase chain reactions indicated that 11 of 12 matured boars were transgene-positive individuals, and that the transgenic vector was most likely disrupted during cloning. Of 11 positive pigs, one (No. 11) lost a part of the terminator region but had the intact promoter and the CDS regions. cDNA sequencing showed that the introduced allele (746G) was expressed in multiple tissues of transgene-positive offspring of No.11. Western blot analysis revealed that BMPR1B protein expression in multiple tissues of transgene-positive F1 piglets was 0.5 to 2-fold higher than that in the transgene-negative siblings. The No. 11 boar showed normal litter size performance as normal pigs from the same breed. Transgene-positive F1 boars produced by No. 11 had higher semen volume, sperm concentration and total sperm per ejaculate than the negative siblings, although the differences did not reached statistical significance. Transgene-positive F1 sows had similar litter size performance to the negative siblings, and more data are needed to adequately assess the litter size performance. In conclusion, we obtained 24 cloned transgenic pigs with the modified porcine BMPR1B CDS using HMC. cDNA sequencing and western blot indicated that the exogenous BMPR1B CDS was successfully expressed in host pigs. The transgenic pigs showed normal litter size performance. However, no significant differences in litter size were found between transgene-positive and negative sows. Our study provides new insight into producing cloned transgenic livestock related to reproductive traits.

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.

In transgenic pig production for generating animal models of human diseases, apoptosis of an early implantation embryo disturbs the transgenic pig production. Porcine embryonic stem cells (pESC) and porcine induced pluripotent stem cells (piPSC) have an advantage for the generation of transgenic pigs; however, porcine stem cells have not yet been established. In addition, epithelial–mesenchymal transition (EMT) may play a critical role in embryo development and apoptosis. Thus, in this study we generated pESC and pIPSC and further examined the changes in EMT and apoptotic markers. We cultured pESC and piPSC in pESC media containing basic fibroblast growth factor (bFGF), doxicyclin, and leukemia inhibitory factor (LIF), and performed RT-PCR and alkaline phosphatase (AP) test to measure pluripotency markers. The RT-PCR results show that OCT-4, NANOG, and SOX2 were expressed in these stem cells, characteristic of stem cells. AP-positive cells were observed in pESCs and piPSC. In addition, we performed immunocytochemistry (ICC) to examine the expression of surface markers, such as SSEA-1 and SSEA-4. We found that pESC and piPSC expressed these markers, indicating that they have a stem cell property similar to rodent and human stem cells. Second, we treated pESC and piPSC with transforming growth factor beta (TGF-β) to examine the relationship between EMT and apoptotic markers, and confirmed a significant variation of EMT and apoptotic markers, i.e. bcl-2, bax, E-cadherin, and vimentin, by Western blot analysis. In a future study, we will examine the effect(s) of various endocrine hormones secreted by the ovary, such as E2 or P4, on the expressions of EMT and apoptotic markers in pESC and piPSC. Consequently, this study will contribute to elucidate underlying mechanism(s) of EMT and apoptosis by endocrine factors to prevent early apoptosis of pig embryos in these porcine stem cells. This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ009599), Rural Development Administration, Republic of Korea.

The presence of multiple copies of porcine endogenous retrovirus (PERV) within the pig genome, and the demonstration that replication competent PERV, that infect human cells in culture, can be isolated from pig cells, directly impacts the drive towards the development of pigs for xenotransplantation. The development of technology to produce pigs that do not propagate PERV has the potential to facilitate the development of xenotransplantation products for human use, and as such, is the focus of this investigation. The shear number of PERV loci, most of which are defective or pseudogenes, renders conventional gene targeting impractical, if not impossible, to inactivate all PERV provirus within the pig genome, including potential replication competent PERV arising from spontaneous recombination. The recently developed RNA interference (RNAi) technology to knockdown/silence post-transcriptional gene expression, offers a promising alternative to achieving this goal. Here, the combination of nuclear transfer cloning and RNAi technology was used to produce pigs that may not propagate PERV. Small interfering RNAs (siRNA) were expressed as short hairpin RNAs (shRNA) against the gag and pol PERV genes, respectively, under the control of a RNA polymerase III (pol III), or a pol II promoter. PERV gag and pol model-genes, in combination with a Green Fluorescent Protein (GFP) reporter system, were developed to assess in vitro PERV target knockdown. Two shRNAs were selected, and transgenic pigs were produced that expressed the anti-gag and -pol shRNAs, in tandem, under the control of a ubiquitous pol II promoter. The anti-gag and -pol shRNAs, effectively knocked down expression of the PERV model-genes, and also endogenous PERV within cells in vitro. PERV knockdown was achieved whether the shRNA was expressed under the control of a RNA pol III, or a pol II promoter. Three litters of cloned pigs were produced. The shRNA construct was expressed by all the transgenic cloned animals, and within all the tissues of transgenic animals tested. PERV expression at the mRNA and PERV particulate levels in the pigs was virtually undetectable, compared with the infectious levels expressed by the positive control PK15 cell line in vitro. Immunofluorescence and Western blotting, with an anti-PERV-envelope antibody, did not detect PERV in pig tissues or cells whether activated or not, as compared to the positive control on PK15 cells. The stable long-term expression of anti-PERV siRNAs was shown to be effective in knocking down PERV expression in cells. However, the very low (sometimes undetectable), and variable levels of expression of PERV in normal pigs make it difficult to obtain suitable control animals for comparison, to assess knockdown of PERV in vivo. This was demonstrated by the observation that even cloned non-transgenic littermates, express levels of PERV as low as that of some of their siRNA transgenic littermates. Further analysis is required to conclusively quantitate in vivo effects in the shRNA transgenic pigs.

Multi-transgenic pigs produced for use in xenotransplantation have to be screened for the presence and expression of porcine endogenous retroviruses (PERV) to select animals with low PERV load. The production of transgenic pigs may also be associated with the integration of the transgene adjacent to or into the locus of a PERV provirus, potentially leading to an enhanced virus expression. Non-transgenic animals, single-transgenic, and multi-transgenic pigs were screened for the presence of PERV-A, -B, and -C and recombinant PERV-A/C using polymerase chain reaction (PCR). PERV expression was determined by real time reverse transcriptase-PCR. An assay based on the activation of PERV in peripheral blood mononuclear cells by mitogens was used to discriminate between low and high PERV producer animals. All animals carried PERV-A and -B. A total of 176 from 181 (97.2%) animals carried PERV-C in the germ line and 18 from 64 animals carried PERV-A/C in the genome of lymphoid cells but not in the germ line. The expression of PERV was very low in all animals and not different between transgenic pigs and non-transgenic animals. PERV expression differed between various pig lines. The highest expression was found in mini-pigs and crossing other pig lines with mini-pigs resulted in increased PERV expression in the progeny. However, expression of viral proteins and particle release were not observed in all transgenic animals. No evidence for elevated PERV expression in (multi-) transgenic pigs was observed. Differences in PERV expression correlated with the genetic background of the animals, not with the specific transgene. Mini-pigs consistently had the highest level of PERV expression and animals with a mini-pig background had a higher level of expression compared with animals without mini-pig background.

We previously reported that transgenic (TG) pigs can be produced from in vitro-matured oocytes using intracytoplasmic sperm injection-mediated gene transfer (ICSI-mediated method) (Kurome et al. 2006 Transgenic Res. 15, 229–240). We subsequently studied the expression of a foreign gene which had been introduced by the ICSI-mediated method. We found that the ICSI-mediated method is considerably less likely than the pronuclear microinjection method to produce embryos in which transgene-positive and transgene-negative cells co-exist, that is, mosaic embryos (Saito et al. 2006 Reprod. Fertil. Dev. 18, 297 abst). Therefore, in order to further investigate the ICSI-mediated method, the present study was conducted to address the integration patterns of foreign genes introduced by this method. In particular, we wished to determine the number of transgene copies and number of chromosomal integration sites. TG pig fetuses, obtained by the ICSI-mediated method in a separate cardiac disease model study, were used in the present study. Porcine cumulus-oocyte complexes that had been collected from slaughterhouse ovaries were subjected to in vitro maturation in NCSU23 medium to produce MII oocytes to be used in this study. Porcine spermatozoa frozen in Beltsville Thawing Solution (BTS) were thawed rapidly in a 37�C water bath, and each spermatozoon was decapitated using ultrasound (28 kHz, 100 W; W-113; Honda Electronics Co., Ltd, Aichi, Japan). The heads (2 to 5 � 105/10 �L) were co-incubated with 2.5 ng �L-1 of rabbit calreticulin cDNA (�MHC-CRT-HA: 7.5 kb) for five min at room temperature, and then microinjected into MII oocytes using a piezo-micromanipulator. An electric stimulus (DC 150 V mm-1, 100 �s) was applied 10 to 40 min after microinjection in order to activate the oocytes. The embryos were cultured in PZM-5 medium for one to two days, and then transferred into the oviducts of recipient gilts, whose estrous cycle had been synchronized using 1000 IU eCG and 1500 IU hCG. Fetuses were collected 33 or 50 days later, and a primary cell line (fibroblast) was established. For each fetus, the number of transgene copies was determined by Southern blot. In addition, the chromosomal sites, where the foreign gene had integrated, were identified, and the number of integration sites was determined by fluoresent in situ hybridization (FISH). A total of 454 ICSI embryos were transferred to 4 recipients (92 to 135 embryos/recipient). All recipients became pregnant and 23 fetuses (5.1%, 23/454), including 7 TG fetuses (30.4%, 7/23), were obtained. Southern blot analysis showed that the number of transgene copies varied between 1 and 300 (1 copy: 1 fetus; 10 copies: 2 fetuses; 30 copies: 3 fetuses; 300 copies: 1 fetus). FISH analysis showed that in TG fetuses, the foreign gene had integrated at only a single chromosomal site, and this site varied from TG fetus to TG fetus. These results demonstrate that, in the case of ICSI-mediated gene transfer, as is the case for gene transfer by pronuclear microinjection, the integration patterns are: multiple copy, random site, and single site integration. This study was supported by PROBRAIN.

Development of efficient strategies for the production of genetically modified pigs

Alzheimer’s disease (AD) is a progressive neurodegenerative disease associated with memory loss and cognitive impairments. An AD transgenic (Tg) pig model would be useful for preclinical testing of therapeutic agents. We generated an AD Tg pig by somatic cell nuclear transfer (SCNT) using a multi-cistronic vector that harbored three AD-related genes with a total of six well-characterized mutations: hAPP (K670N/M671L, I716V, and V717I), hTau (P301L), and hPS1 (M146V and L286P). Four AD Tg cell lines were established from Jeju black pig ear fibroblasts (JB-PEFs); the resultant JB-PEFAD cells harbored transgene integration, expressed transgene mRNAs, and had normal karyotypes. Tg line #2–1, which expressed high levels of the transgenes, was used for SCNT; cleavage and blastocyst rates of embryos derived from this line were lower than those of Non-Tg. These embryos yielded three piglets (Jeju National University AD-Tg pigs, JNUPIGs) revealed by microsatellite testing to be genetically identical to JB-PEFAD. Transgenes were expressed in multiple tissues, and at especially high levels in brain, and Aβ-40/42, total Tau, and GFAP levels were high in brains of the Tg animals. Five or more copies of transgenes were inserted into chromosome X. This is the first report of an AD Tg pig derived from a multi-cistronic vector.

Production of transgenic livestock by pronuclear microinjection of DNA into fertilized zygotes suffers from the compounded inefficiencies of low embryo survival and low integration frequencies of the injected DNA into the genome. These inefficiencies are one of the major obstacles to the large-scale use of pronuclear microinjection techniques in livestock. We investigated exploiting the properties of recombinase proteins that allow them to bind DNA to generate transgenic animals via pronuclear microinjection. In theory, the use of recombinase proteins has the potential to generate transgenic animals with targeted changes, but in practice we found that the use of RecA recombinase-coated DNA increases the efficiency of transgenic livestock production. The use of RecA protein resulted in a significant increase in both embryo survival rates and transgene integration frequencies. Embryo survival rates were doubled in goats, and transgene integration was 11-fold higher in goats and three-fold higher in pigs when RecA protein-coated DNA was used compared with conventional DNA constructs without RecA protein coating. However, a large number of the transgenic founders generated with RecA protein-coated DNA were mosaic. The RecA protein coating of DNA is straightforward and can be applied to any species and any existing microinjection apparatus. These findings represent significant improvements on standard pronuclear microinjection methods by enabling the more efficient production of transgenic livestock.