[Progress on spermatogonial stem cells of large animals].

Spermatogonial stem cells (SSCs) are male germline stem cells that reside in the basement membrane of the seminiferous tubule in the testis. SSCs are characterized by their capability of self-renewal to maintain the stem cell pool throughout the lifespan and commitments to germ line after puberty, thus transmitting the genetic information from parents to the SSC-derived progenies. SSCs can be isolated from testis, propagated in vitro, and induced to differentiate into varied germ cells. Although significant progress has been made in the field of rodent SSCs, the SSCs of large animals have advanced slowly. Studies on SSCs of large animal models can offer insights into the physiological and pathological mechanism of human reproduction. Moreover, SSCs of agricultural large animals can be used as an essential tool for multiplication of elite animal individuals, and generation of genetically modified livestock with valuable economic traits. In this review, we summarize the recent progress on SSCs of large animal models for agricultural and medical purposes, and discuss the present problems and future prospects. This review can give an overall view of large animal SSCs as respect to their applications in novel alternative reproductive technologies, generation of transgenic animals, treatment of male infertility and regenerative medicine.

[Progress and application of genome-edited pigs in biomedical research].

Genome editing technologies (GETs) can precisely alter the genomic sequences and modify the genetic information at the target site of an organism. Since the beginning of the 21st century, the GETs, including zinc finger nucleases (ZFN), transcription-activating-like receptor factor (TALEN), and clustered regularly interspaced short palindromic repeats/Cas endonucleases (CRISPR/Cas), have been successively developed. The GETs can easily engineer the targeted genomic site of animals to exhibit a desired phenotype(s), thereby providing valuable tools in biomedical research. The pigs are closely related to human, in terms of similarities in physiological properties and pathogenic characters. Thus, pigs have been used as important animal models in studies of human disease, xenotransplantation, and humanized organs regeneration. In this review, we summarize the development of the three GETs, research progress of genome-edited pigs as disease models and organ donors for xenotransplantation, and the prospects of their applications in future biomedical research.

Advances in site-specific integration of transgene in animal genome.

The traditional transgenic technologies, such as embryo microinjection, transposon-mediated integration, or lentiviral transfection, usually result in random insertions of the foreign DNA into the host genome, which could have various disadvantages in the establishment of transgenic animals. Therefore, a strategy for site-specific integration of a transgene is needed to generate genetically modified animals with accurate and identical genotypes. However, the efficiency for site-specific integration of transgene is very low, which is mainly caused by two issues. The first one is the low efficiency of inducing double-strand break (DSB) at the target site of host genome in the initial process. The second one is the low efficiency of homologous recombination repair (HDR) between the target site and the donor plasmid carrying homologous arm and foreign genes. HDR is the most common mechanism for site-specific integration of a transgene. DSBs can stimulate DNA repair mainly by two competitive mechanisms, HDR and nonhomologous end joining (NHEJ). Hence, activation of HDR or inhibition of NHEJ can promote the HDR in the integration processes, thereby optimizing a specific targeting of the transgene. In this review, we summarize the recent advances in strategies for improving the site-specific integration of foreign transgene in transgenic technologies.

Establishment of porcine Xist knockout model using CRISPR/Cas9 system.

Somatic cell nuclear transfer technique has great applications in livestock breeding, production of genetically modified animals, rescue of endangered species and treatment of human diseases. However, the currently low efficiency in animals cloning, an average of less than 5%, greatly hindered the rapid development of this technique. Among many factors which affect the efficiency of cloning pigs, X chromosome inactivation is an important one. Moreover, Xist gene is closely related to X chromosome inactivation, suggesting that it may directly or indirectly affects cloning efficiency. In this study, multiple sgRNAs were designed based on the CRISPR/Cas system, and two sites (Target 3 and Target 4) whose mutation efficiency were 1% and 3% at the cellular level were selected. We successfully knocked out Xist with 100% efficiency by microinjecting sgRNAs for Target 3 and Target 4 in embryo. Finally, 6 cloning piglets were born including two Xist-fully-knockout piglets. The follow-up studies on increasing cloning efficiency can be carried out based on the Xist-knockout model.

Transgenic expression of human cytoxic T-lymphocyte associated antigen4-immunoglobulin (hCTLA4Ig) by porcine skin for xenogeneic skin grafting.

Porcine skin is frequently used as a substitute of human skin to cover large wounds in clinic practice of wound care. In our previous work, we found that transgenic expression of human cytoxicT-lymphocyte associated antigen4-immunoglobulin (hCTLA4Ig) in murine skin graft remarkably prolonged its survival in xenogeneic wounds without extensive immunosuppression in recipients, suggesting that transgenic hCTLA4Ig expression in skin graft may be an effective and safe method to prolong xenogeneic skin graft survival. In this work, using a transgene construct containing hCTLA4Ig coding sequence under the drive of human Keratine 14 (k14) promoter, hCTLA4Ig transgenic pigs were generated by somatic nuclear transfer. The derived transgenic pigs were healthy and exhibited no signs of susceptibility to infection. The hCTLA4Ig transgene was stably transmitted through germline over generations, and thereby a transgenic pig colony was established. In the derived transgenic pigs, hCTLA4Ig expression in skin was shown to be genetically stable over generations, and detected in heart, kidney and corneal as well as in skin. Transgenic hCTLA4Ig protein in pigs exhibited expected biological activity as it suppressed human lymphocyte proliferation in human mixed lymphocyte culture to extents comparable to those of commercially purchased purified hCTLA4Ig protein. In skin grafting from pigs to rats, transgenic porcine skin grafts exhibited remarkably prolonged survival compared to the wild-type skin grafts derived from the same pig strain (13.33 ± 3.64 vs. 6.25 ± 2.49 days, P < 0.01), further indicating that the transgenic hCTLA4Ig protein was biologically active and capable of extending porcine skin graft survival in xenogeneic wounds. The transgenic pigs generated in this work can be used as a reproducible resource to provide porcine skin grafts with extended survival for wound coverage, and also as donors to investigate the impacts of hCTLA4Ig on xenotransplantation of other organs (heart, kidney and corneal) due to the ectopic transgenic hCTLA4Ig expression.

Space-bound optical source for satellite-ground decoy-state quantum key distribution.

Satellite-ground quantum key distribution has embarked on the stage of engineering implementation, and a global quantum-secured network is imminent in the foreseeable future. As one payload of the quantum-science satellite which will be ready before the end of 2015, we report our recent work of the space-bound decoy-state optical source. Specialized 850 nm laser diodes have been manufactured and the integrated optical source has gotten accomplished based on these LDs. The weak coherent pulses produced by our optical source feature a high clock rate of 100 MHz, intensity stability of 99.5%, high polarization fidelity of 99.7% and phase randomization. A series of space environment tests have been conducted to verify the optical source's performance and the results are satisfactory. The emulated final secure keys are about 120 kbits during one usable pass of the low Earth orbit satellite. This work takes a significant step forward towards satellite-ground QKD and the global quantum-secured network.

[Plasma concentrations of vascular endothelial growth factor and tissue factor in children with acute lymphoblastic leukemia].

OBJECTIVE: To detect plasma concentrations of vascular endothelial cell growth factor (VEGF) and tissue factor (TF) in children with acute lymphoblastic leukemia (ALL) and explore their clinical significance in ALL. METHODS: Thirty-three children with newly diagnosed ALL, including 18 cases of low risk, 7 cases of moderate risk and 8 cases of high risk, were enrolled in this study. Twenty-five patients received a complete remission and 8 cases were in non-remission after conventional remission induction chemotherapy. Plasma concentrations of VEGF and TF in the patients were detected using ELISA before and after treatment. Sixteen healthy children served as normal control group. RESULTS: Plasma concentrations of VEGF and TF in ALL patients before treatment were significantly higher than those in normal controls (P < 0.01). Plasma concentrations of VEGF and TF in the non-remission group before treatment were significantly higher than those in the remission group (P < 0.05) and the control group (P < 0.01). After treatment the plasma concentrations of VEGF and TF in the non-remission group were not significantly reduced and higher than those in the remission and the control groups (P < 0.01). There were significant differences in plasma concentrations of VEGF and TF among the low-risk, moderate-risk and high-risk groups before and after treatment (P < 0.05). Plasma concentrations of VEGF and TF in the high risk group were not significantly reduced after treatment and higher than those in the control group (P < 0.01). A linear correlation was noted between plasma VEGF and TF concentrations in ALL patients before treatment (r=0.50, P < 0.01). CONCLUSIONS: VEGF and TF play an important role in the development of ALL and may be useful to the evaluation of the severity and the outcome in ALL.

[Expression of survivin and P63 protein in B cell non-Hodgkin's lymphoma and their effects on cell apoptosis and proliferation].

The study was aimed to explore the possible roles of survivin and P63 protein in the development and progression of B cell non-Hodgkin's lymphoma (B-NHL) and their relation with cell apoptosis and proliferation. TdT-mediated dUTP nick end labeling (TUNEL) and immunohistochemistry were used to detect the survivin and P63 protein expression, cell apoptosis and proliferating cell nuclear antigen (PCNA) level in 43 cases of B-NHL and 10 cases of reactive hyperplasia lymphoid (RHL) tissues. The results indicated that the positive rates of survivin and P63 protein expression were 69.8% (30/43) and 82.7% (30/43) respectively. The expression of survivin and P63 protein was 10% (1/10) and 40% (4/10) in RHL tissues of 10 cases. The expression of survivin in aggression B-NHL was higher than that in indolent B-NHL (83.3% vs 46.2%, P < 0.01). The expression of P63 proteins in aggressive B-NHL was higher than that in indolent B-NHL (86.7% vs 76.9%, P > 0.05). Apoptotic index (AI) and proliferation index (PI) correlated positively with expression of survivin (r = 0.429, P < 0.01; r = 0.348, P < 0.01), and so do with expression of P63 proteins (r = 0.451, P < 0.01; r = 0.369, P < 0.05). In addition, AI and PI were positively related (r = 0.598, P < 0.001). It is concluded that survivin may participate in the regulation mechanism of B-NHL cell apoptosis and proliferation, P63 as an oncogene enhances proliferation and takes part in the development of B-NHL. There may be a close relationship between survivin and P63 protein in the regulation of lymphocyte proliferative kinetics.

[A study on the expression of myeloid cell leukemia 1 proteins and survivin and their relation with B cell apoptosis in non-Hodgkin's lymphoma].

OBJECTIVE: To explore the expression of myeloid cell leukemia 1 (MCL-1) proteins and survivin and its correlation with cell apoptosis as well as with the development and progression of B cell non-Hodgkin's lymphoma (B-NHL). METHODS: TdT-mediated dUTP nick end labeling and immunohistochemistry were used to study cell apoptosis and expression of MCL-1 proteins and survivin proteins in 43 patients with B-NHL and 10 with reactive hyperplasia (RH) lymphoid tissue. RESULTS: The positive rate of MCL-1 proteins and survivin proteins was 58.1% (25/43) and 69.8% (30/43) respectively. The expression of MCL-1 proteins was not detected in RH lymphoid tissue, but that of survivin was detected in 10.0% (1/10). The expression of MCL-1 proteins in aggressive B-NHL was higher than that in indolent B-NHL (70.0 % vs 30.8 %, P < 0.05). The expression of survivin in aggressive B-NHL was also higher than that in indolent B-NHL (80.0% vs 46.2%, P < 0.05). Apoptotic index (AI) was not correlated positively with the expression of MCL-1, but correlated positively with the expression of survivin (r = 0.429, P < 0.01). MCL-1 and survivin were correlated positively in B-NHL (r = 0.598, P < 0.001). CONCLUSIONS: MCL-1 proteins as family member of BCL-2 have no influence on apoptosis but survivin may participate in the regulation mechanism of B-NHL apoptosis. It is indicated that the two proteins with a close relationship may take part in the development and progression of B-NHL.