Effects of sperm pretreatment on efficiency of ICSI-mediated gene transfer in pigs

Intracytoplasmic sperm injection (ICSI)-mediated gene transfer has recently been shown to be an effective technique for producing transgenic pigs; however, the types of sperm pretreatment having the most beneficial effects on post-ICSI embryogenesis or transgenic efficiency have not been clarified. In the present study, we performed ICSI-mediated gene transfer using pig sperm subjected to various pretreatments and determined the developmental potential of sperm-injected oocytes and introduction efficiency of exogenous DNA. Embryos were then transferred to recipient pigs to confirm gene transfer efficiency during the fetal period. When ICSI was performed using unfrozen sperm heads with tails removed by piezo-pulse, the rates of blastocyst formation (14.2%, 17/120) and transgene (EGFP) expression (11.8%, 2/17) were both low. When unfrozen sperm heads were used that were removed by sonication, EGFP expression efficiency (11/21, 52.4%) improved significantly (P<0.05). Pretreatment of unfrozen sperm with a surfactant or acrosomal reaction did not further improve the rates of blastocyst formation and EGFP expression. However, use of the heads of sperm frozen-thawed with or without a cryoprotective agent resulted in rates of blastocyst formation and EGFP expression that tended to be generally high (23.0%, 14/61-33.8%, 26/77 and 42.9%, 6/14-66.7%, 10/15). A total of 219 in vitro matured oocytes were fertilized by ICSI-mediated gene transfer using the heads of frozen-thawed sperm and then transferred into two recipient pigs. Seven fetuses were obtained, and EGFP expression and integration of the transgene (10-30 copies) were confirmed in two of the seven fetuses. Use of unfrozen sperm thus confers no advantages on ICSI-mediated gene transfer, and although further investigations are needed, frozen-thawed sperm heads appear to be useful in ICSI-mediated gene transfer.     

Production of transgenic and non-transgenic clones in miniature pigs by somatic cell nuclear transfer

Miniature pigs have been recognized as valuable experimental animals in various fields such as medical and pharmaceutical research. However, the amount of information on somatic cell cloning in miniature pigs, as well as genetically modified miniature pigs, is much less than that available for common domestic pigs. The objective of the present study was to establish an efficient technique of cloning miniature pigs by somatic cell nuclear transfer. A high pregnancy rate was achieved following transfer of parthenogenetic (3/3) and cloned (5/6) embryos using female miniature pigs in the early pregnancy period as recipients after estrus synchronization with prostaglandin F2 alpha analog and gonadotrophins. The production efficiency of the cloned miniature pigs using male and female fetal fibroblasts as nucleus donors was 0.9% (2/215 and 3/331, respectively). Cloned miniature pigs were also produced efficiently (7.8%, 5/64) by transferring reconstructed embryos into the uteri of common domestic pigs. When donor cells transfected with the green fluorescent protein (GFP) gene were used in nuclear transfer, the production efficiency of the reconstructed embryos and rate of blastocyst development were comparable to those obtained by non-transfected cells. When transfected cell-derived reconstructed embryos were transferred to three common domestic pig recipients, all became pregnant, and a total of ten transgenic cloned miniature pigs were obtained (piglet production efficiency: 2.7%, 10/365). Hence, we were able to establish a practical system for producing cloned and transgenic-cloned miniature pigs with a syngeneic background.     

In vitro production of porcine embryos: current status,future perspectives and alternative applications

The pig is considered to be a suitable source of cells and organs for xenotransplants, as well as a transgenic animal to produce specific proteins, given the biological similarities it shares with human beings. However, the in vitro embryo production system in pigs is inefficient compared with those in other mammals, such as cattle or mice. Although numerous modifications have been applied to improve the efficiency of in vitro embryo production systems in pigs, not much progress has been made to overcome the problem of polyspermy, and low developmental ability due to insufficient cytoplasmic abilities of in vitro matured oocytes and improper culture conditions for the in vitro produced embryos. Recent achievements, such as the establishment of chemically defined medium and utilization of ‘zona hardening’ technique, have gained some success. However, further research for the reduction of polyspermy and detrimental effects of the culture systems in pigs is still needed.     

Production of Diabetic Offspring Using Cryopreserved Epididymal Sperm by In Vitro Fertilization and Intrafallopian Insemination Techniques in Transgenic Pigs

Somatic cell nuclear transfer (SCNT) is a useful technique for creating pig strains that model human diseases. However, production of numerous cloned disease model pigs by SCNT for large-scale experiments is impractical due to its complexity and inefficiency. In the present study, we aimed to establish an efficient procedure for proliferating the diabetes model pig carrying the mutant human hepatocyte nuclear factor-1α gene. A founder diabetes transgenic cloned pig was generated by SCNT and treated with insulin to allow for normal growth to maturity, at which point epididymal sperm could be collected for cryopreservation. In vitro fertilization and intrafallopian insemination using the cryopreserved epididymal sperm resulted in diabetes model transgenic offspring. These results suggest that artificial reproductive technology using cryopreserved epididymal sperm could be a practical option for proliferation of genetically modified disease model pigs.     

Transgenic mouse offspring generated by ROSI

The production of transgenic animals is an important tool for experimental and applied biology. Over the years, many approaches for the production of transgenic animals have been tried, including pronuclear microinjection, sperm-mediated gene transfer, transfection of male germ cells, somatic cell nuclear transfer and the use of lentiviral vectors. In the present study, we developed a new transgene delivery approach, and we report for the first time the production of transgenic animals by co-injection of DNA and round spermatid nuclei into non-fertilized mouse oocytes (ROSI). The transgene used was a construct containing the human CMV immediate early promoter and the enhanced GFP gene. With this procedure, 12% of the live offspring we obtained carried the transgene. This efficiency of transgenic production by ROSI was similar to the efficiency by pronuclear injection or intracytoplasmic injection of male gamete nuclei (ICSI). However, ICSI required fewer embryos to produce the same number of transgenic animals. The expression of Egfp mRNA and fluorescence of EGFP were found in the majority of the organs examined in 4 transgenic lines generated by ROSI. Tissue morphology and transgene expression were not distinguishable between transgenic animals produced by ROSI or pronuclear injection. Furthermore, our results are of particular interest because they indicate that the transgene incorporation mediated by intracytoplasmic injection of male gamete nuclei is not an exclusive property of mature sperm cell nuclei with compact chromatin but it can be accomplished with immature sperm cell nuclei with decondensed chromatin as well. The present study also provides alternative procedures for transgene delivery into embryos or reconstituted oocytes.     

Characterization of Transgenic Pigs That Express Human Decay Accelerating Factor and Cell Membrane‐tethered Human Tissue Factor Pathway Inhibitor

The expression of human complement regulatory proteins (hCRP; hDAF, hCD59, and hMCP) in pig tissues has been suggested as one of strategies to overcome the hyperacute rejection (HAR) in pig‐to‐human transplantation. Expression of human tissue factor pathway inhibitor (hTFPI) in porcine endothelial cells has been suggested as a remedy to overcome microvascular thrombosis. To investigate the effects of these combined transgenes, we established transformed pig cells expressing human decay accelerating factor (hDAF) under the control of enhancer promoter (5′LTR‐PCMVIE), and the fusion protein (hTFPI/hCD4) consisting of the functional domains (K1 and K2) of hTFPI and membrane‐tethering domains (D3 and D4) of hCD4 under the control of PCMVIE. Transgenic pigs were generated with the transformed porcine cells through somatic cell nuclear transfer (SCNT) technology. Analysis of quantitative PCR and real‐time quantitative RT‐PCR showed that four copies of hDAF were integrated and 391 copies of hDAF mRNA expressed in the cells of the transgenic pig. The enhancing activity of 5′LTR was approximately 2 fold compared to CMVIE promoter only. The cell viability test showed that more than 80% of ear cells were viable in the presence of 50% human serum. The chromogenic substrate assay and immunocytochemical staining with tail cells showed that the TFPI activity of fusion protein was observed on the cell membrane. The membrane localization of hDAF and hTFPI proteins was observed by immunocytochemical staining, and the expression of transgenes in heart and liver tissues was also confirmed by immunohistochemistry.     

Factors affecting porcine sperm mediated gene transfer

“Sperm mediated gene transfer” (SMGT) is based on the ability of sperm cells to bind exogenous DNA. The main objective of this study was to improve the production of transgenic pigs by SMGT. Taking into account that there is a lack of repeatability in studies of SMGT and that the mechanism of binding and internalization of exogenous DNA is a question that has not been solved, different factors involved in the production of transgenic animals by SMGT method were evaluated. Here we set out to: (1) evaluate the sperm capacity to bind exogenous DNA after DMSO treatment; (2) determine the location of the transgene–spermatozoa interaction; and (3) evaluate the efficiency of production of transgenic piglets by deep intrauterine artificial insemination (AI) with sperm incubated with DNA. The percentage of DNA binding was higher than 30% after 2 h of co-culture, but it was not affected by sperm treatment with DMSO (0.3% or 3%). The integrity of the sperm plasma membrane plays a critical role in DNA interaction, and altered plasma membranes facilitate interactions with exogenous DNA. DNA bound mainly to spermatozoa with reduced viability. DNA molecules were found to be mainly associated to the post-acrosomal region (61.9%). After deep intrauterine AI a total of 29 piglets were obtained, but none of them integrated the transgene. In conclusion, although it has been confirmed that DNA can associate with boar spermatozoa, the efficiency of producing transgenic pigs by AI was not confirmed by the present experiments, mainly due to a reduced DNA binding to functional spermatozoa.     

Technical development for production of gene-modified laboratory rats

Transgenic rats have been used as model animals for human diseases and organ transplantation and as animal bioreactors for protein production. In general, transgenic rats are produced by pronuclear microinjection of exogenous DNA. Improvement of post-injection survival has been achieved by micro-vibration of the injection pipette. The promoter region, structural gene, chain length and strand ends of the exogenous DNA are not involved in the production efficiency of transgenic rats. Exogenous DNA prepared at 5 microg/ml seemed to be better integrated than lower and higher concentrations. Intracytoplasmic sperm injection (ICSI) has been successfully achieved in rats using a piezo-driven injection pipette. The ICSI technique has not only been applied to rescue infertile male strains but also to produce transgenic rats. The optimal DNA concentration for the ICSI-tg method (0.1 to 0.5 microg/ml) is lower than that for the conventional pronuclear microinjection. Production efficiency was improved when the membrane structure of the sperm head was partially disrupted by detergent or ultrasonic treatment before exposure to the exogenous DNA solution. For successful production of transgenic rats with a modified endogenous gene, establishment of embryonic stem cell lines or alternatively male germline stem cell lines and technical development of somatic cell nuclear transfer are still necessary for this species.     

Intrapancreatic ectopic splenic tissue found in a cloned miniature pig

Somatic cell nuclear transfer (SCNT) is a cost-effective technique for producing transgenic pigs. However, abnormalities in the cloned pigs might prevent use these animals for clinical applications or disease modeling. In the present study, we generated several cloned pigs. One of the pigs was found to have intrapancreatic ectopic splenic tissue during histopathology analysis although this animal was grossly normal and genetically identical to the other cloned pigs. Ectopic splenic tissue in the pancreas is very rare, especially in animals. To the best of our knowledge, this is the first such report for cloned pigs.     

Development of genome engineering technologies in cattle: from random to specific

The production of transgenic farm animals(e.g., cattle) via genome engineering for the gain or loss of gene functions is an important undertaking. In the initial stages of genome engineering, DNA micro-injection into one-cell stage embryos(zygotes) followed by embryo transfer into a recipient was performed because of the ease of the procedure.However, as this approach resulted in severe mosaicism and has a low efficiency, it is not typically employed in the cattle as priority, unlike in mice. To overcome the above issue with micro-injection in cattle, somatic cell nuclear transfer(SCNT) was introduced and successfully used to produce cloned livestock. The application of SCNT for the production of transgenic livestock represents a significant advancement, but its development speed is relatively slow because of abnormal reprogramming and low gene targeting efficiency. Recent genome editing technologies(e.g.,ZFN, TALEN, and CRISPR-Cas9) have been rapidly adapted for applications in cattle and great results have been achieved in several fields such as disease models and bioreactors. In the future, genome engineering technologies wil accelerate our understanding of genetic traits in bovine and wil be readily adapted for bio-medical applications in cattle.     

Genetic engineering of mammalian embryos

A gene consisting of the mouse metallothionein I promoter/regulator (MT) fused to the human growth hormone (hGH) structural gene (MThGH) was microinjected into rabbit, sheep and pig eggs. Visualization of nuclear structures was accomplished by interference-contrast (I-C) microscopy for rabbit and sheep eggs and by centrifugation and I-C microscopy for pig eggs. The gene integrated into the chromosomes of each species with an efficiency of 13% in rabbits, 1% in sheep and 10% in pigs. Human GH mRNA was detected in the liver of transgenic rabbits as well as tail and ear samples of pigs. Immunoreactive hGH was present in the serum of a transgenic rabbit and plasma of most transgenic pigs. In several pigs hGH levels increased between birth and 90 d of age. The presence of substantial quantities of hGH in plasma of pigs did not increase postnatal somatic growth rates. Founder animals will be bred and their transgenic and control progeny used to assess the effects of hGH on feed efficiency and carcass composition. These experiments demonstrate the feasibility of introducing foreign genes into the genome of several animal species by microinjection of eggs.     

Generation of RUNX3 knockout pigs using CRISPR/Cas9‐mediated gene targeting

Pigs are an attractive animal model to study the progression of cancer because of their anatomical and physiological similarities to human. However, the use of pig models for cancer research has been limited by availability of genetically engineered pigs which can recapitulate human cancer progression. Utilizing genome editing technologies such as CRISPR/Cas9 system allows us to generate genetically engineered pigs at a higher efficiency. In this study, specific CRISPR/Cas9 systems were used to target RUNX3, a known tumour suppressor gene, to generate a pig model that can induce gastric cancer in human. First, RUNX3 knockout cell lines carrying genetic modification (monoallelic or biallelic) of RUNX3 were generated by introducing engineered CRISPR/Cas9 system specific to RUNX3 into foetal fibroblast cells. Then, the genetically modified foetal fibroblast cells were used as donor cells for somatic cell nuclear transfer, followed by embryo transfer. We successfully obtained four live RUNX3 knockout piglets from two surrogates. The piglets showed the lack of RUNX3 protein in their internal organ system. Our results demonstrate that the CRISPR/Cas9 system is effective in inducing mutations on a specific locus of genome and the RUNX3 knockout pigs can be useful resources for human cancer research and to develop novel cancer therapies.     

体细胞克隆太湖猪出生

以中国太湖猪胎儿成纤维细胞为核供体,以体外成熟卵母细胞为核受体,以杜洛克猪为代孕母猪,进行了太湖猪体细胞克隆技术的研究,成功地获得了首例体细胞克隆太湖猪仔猪.经微卫星DNA多态性鉴定,确定克隆猪来自供核细胞,与代孕母猪无亲缘关系.本研究将为体细胞克隆技术在我国太湖猪育种、建立人类疾病模型等研究方面提供可行的技术方法.     

Use of Transgenic Animals to Improve Human Health and Animal Production

Contents Transgenic animals are more widely used for various purposes. Applications of animal transgenesis may be divided into three major categories: (i) to obtain information on gene function and regulation as well as on human diseases, (ii) to obtain high value products (recombinant pharmaceutical proteins and xeno-organs for humans) to be used for human therapy, and (iii) to improve animal products for human consumption. All these applications are directly or not related to human health. Animal transgenesis started in 1980. Important improvement of the methods has been made and are still being achieved to reduce cost as well as killing of animals and to improve the relevance of the models. This includes gene transfer and design of reliable vectors for transgene expression. This review describes the state of the art of animal transgenesis from a technical point of view. It also reports some of the applications in the medical field based on the use of transgenic animal models. The advance in the generation of pigs to be used as the source of organs for patients and in the preparation of pharmaceutical proteins from milk and other possible biological fluids from transgenic animals is described. The projects in course aiming at improving animal production by transgenesis are also depicted. Some the specific biosafety and bioethical problems raised by the different applications of transgenesis, including consumption of transgenic animal products are discussed.     

Progress on gene transfer in farm animals

Transgenic pigs and sheep have been produced by the microinjection of single-cell zygotes and two-cell ova with linear molecules of mouse metallothionein I (MT) promoter/regulator fused to either the human growth hormone (hGH) or bovine growth hormone (bGH) structural genes. The foreign genes integrated into the chromosomes of 3 of 111 lambs or fetuses and 31 of 341 pigs or fetuses examined. Immunoreactive hGH or bGH was present in the plasma of two transgenic lambs and 19 transgenic pigs. The hGH concentration in plasma varied greatly among pigs and was unrelated to the number of gene copies that had integrated. Rate of growth was not enhanced in any of the transgenic pigs in comparison to their littermate controls. However, bGH and hGH exerted definite biological effects in transgenic pigs as evidenced by significantly depressed backfat measurements, elevated levels of insulin-like growth factor (IGF-I), stimulation of mammary development (by hGH) and reduction in porcine growth hormone (pGH) to nondetectable levels in plasma. Five of six founder transgenic pigs transmitted the MT-hGH gene construct to one or more progeny. Three progeny of a boar that expressed hGH also expressed the foreign gene.     

Inability of heterologous growth hormone (GH) to regulate GH binding protein in GH-transgenic swine

Transgenic pigs expressing bovine, ovine, or human growth hormone (GH) structural genes fused to mouse metallothionein-I (mMT-bGH), ovine MT (oMT-oGH), or mouse transferrin (mTf-hGH) promoters were used to study the effects of GH on the regulation of serum GH-binding protein (GHBP). In the 14 transgenic pigs studied, circulating concentrations of heterologous GH ranged from 15 to 2,750 ng/mL. Using chromatographic methods, specific binding of GH was detected in serum from normal pigs but was undetectable in serum from all the transgenic pigs used, probably as a result of the high serum concentrations of heterologous GH present in these animals. Thus, to avoid interference of binding by high GH concentrations, serum samples were subjected to immunoblotting using a specific anti-GHBP antibody. A specific 54-kDa band was detected in normal pig serum as well as in sera from mMT-bGH, oMT-oGH, and mTf-hGH pigs. Additionally, sera from transgenic mMT-bGH pigs and their sibling controls were subjected to immunoprecipitation with an anti-GHBP antibody followed by immunoblotting with the same antibody. With this technique, we detected two specific bands of 53 and 45 kDa that could represent different degrees of glycosylation of GHBP. As determined by densitometric analysis the amount of GHBP in transgenic pig sera was similar to that detected in sera of the respective control animals. The amount of circulating GHBP remained unchanged even in oMT-oGH and mTf-hGH pigs that were exposed from birth to circulating concentrations of GH as high as 2,750 ng/mL. Thus, we conclude that heterologous GH do not act as modulators ofthe serum GHBP in pigs.     

Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum

Monomeric Plum (Plum), a far-red fluorescent protein with photostability and photopermeability, is potentially suitable for in vivo imaging and detection of fluorescence in body tissues. The aim of this study was to generate transgenic cloned pigs exhibiting systemic expression of Plum using somatic cell nuclear transfer (SCNT) technology. Nuclear donor cells for SCNT were obtained by introducing a Plum-expression vector driven by a combination of the cytomegalovirus early enhancer and chicken beta-actin promoter into porcine fetal fibroblasts (PFFs). The cleavage and blastocyst formation rates of reconstructed SCNT embryos were 81.0% (34/42) and 78.6% (33/42), respectively. At 36–37 days of gestation, three fetuses systemically expressing Plum were obtained from one recipient to which 103 SCNT embryos were transferred (3/103, 2.9%). For generation of offspring expressing Plum, rejuvenated PFFs were established from one cloned fetus and used as nuclear donor cells. Four cloned offspring and one stillborn cloned offspring were produced from one recipient to which 117 SCNT embryos were transferred (5/117, 4.3%). All offspring exhibited high levels of Plum fluorescence in blood cells, such as lymphocytes, monocytes and granulocytes. In addition, the skin, heart, kidney, pancreas, liver and spleen also exhibited Plum expression. These observations demonstrated that transfer of the Plum gene did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of transgenic cloned pigs that systemically expressed Plum. This is the first report of the generation and characterization of transgenic cloned pigs expressing the far-red fluorescent protein Plum.     

Factors affecting porcine sperm mediated gene transfer

“Sperm mediated gene transfer” (SMGT) is based on the ability of sperm cells to bind exogenous DNA. The main objective of this study was to improve the production of transgenic pigs by SMGT. Taking into account that there is a lack of repeatability in studies of SMGT and that the mechanism of binding and internalization of exogenous DNA is a question that has not been solved, different factors involved in the production of transgenic animals by SMGT method were evaluated. Here we set out to: (1) evaluate the sperm capacity to bind exogenous DNA after DMSO treatment; (2) determine the location of the transgene–spermatozoa interaction; and (3) evaluate the efficiency of production of transgenic piglets by deep intrauterine artificial insemination (AI) with sperm incubated with DNA. The percentage of DNA binding was higher than 30% after 2 h of co-culture, but it was not affected by sperm treatment with DMSO (0.3% or 3%). The integrity of the sperm plasma membrane plays a critical role in DNA interaction, and altered plasma membranes facilitate interactions with exogenous DNA. DNA bound mainly to spermatozoa with reduced viability. DNA molecules were found to be mainly associated to the post-acrosomal region (61.9%). After deep intrauterine AI a total of 29 piglets were obtained, but none of them integrated the transgene. In conclusion, although it has been confirmed that DNA can associate with boar spermatozoa, the efficiency of producing transgenic pigs by AI was not confirmed by the present experiments, mainly due to a reduced DNA binding to functional spermatozoa.     

Improved development of somatic cell cloned bovine embryos by a mammary gland epithelia cells in vitro model

Previous studies have established a bovine mammary gland epithelia cells in vitro model by the adenovirus-mediated telomerase (hTERT-bMGEs). The present study was conducted to confirm whether hTERT-bMGEs were effective target cells to improve the efficiency of transgenic expression and somatic cell nuclear transfer (SCNT). To accomplish this, a mammary-specific vector encoding human lysozyme and green fluorescent protein was used to verify the transgenic efficiency of hTERT-bMGEs, and untreated bovine mammary gland epithelial cells (bMGEs) were used as a control group. The results showed that the hTERT-bMGEs group had much higher transgenic efficiency and protein expression than the bMGEs group. Furthermore, the nontransgenic and transgenic hTERT-bMGEs were used as donor cells to evaluate the efficiency of SCNT. There were no significant differences in rates of cleavage or blastocysts or hatched blastocysts of cloned embryos from nontransgenic hTERT-bMGEs at passage 18 and 28 groups (82.8% vs. 81.9%, 28.6% vs. 24.8%, 58.6% vs. 55.3%, respectively) and the transgenic group (80.8%, 26.5% and 53.4%); however, they were significantly higher than the bMGEs group (71.2%, 12.8% and 14.8%), (p < 0.05). We confirmed that hTERT-bMGEs could serve as effective target cells for improving development of somatic cell cloned cattle embryos.