hNTCP­expressing primary pig hepatocytes are a valuable tool for investigating hepatitis B virus infection and antiviral drugs.

Primary human hepatocytes (PHHs) are the 'gold standard' for investigating hepatitis B virus (HBV) infection and antiviral drugs. However, poor availability, variation between batches and ethical issues regarding PHHs limit their applications. The discovery of human sodium taurocholate co­transporting polypeptide (hNTCP) as a functional HBV receptor has enabled the development of a surrogate model to supplement the use of PHHs. In the present study, the evolutionary distance of seven species was assessed based on single­copy homologous genes. Based on the evolutionary distance and availability, PHHs and primary rabbit hepatocytes (PRHs) were isolated and infected with hNTCP­recombinant lentivirus, and susceptibility to HBV infection in the two cell types was tested and compared. In addition, HBV infection efficiency of hNTCP­expressing PPHs with pooled HBV­positive serum and purified particles was determined. The potential use of HBV­infected hNTCP­expressing PPHs for drug screening was assessed. The results demonstrated that pigs and rabbits are closer to humans in the divergence tree compared with mice and rats, indicating that pigs and rabbits were more likely to facilitate the HBV post­entry lifecycle. Following hNTCP complementation and HBV infection, PPHs and Huh7D human hepatocellular carcinoma cells, but not PRHs, exhibited increased hepatitis B surface antigen and hepatitis B e­antigen secretion, covalently closed circular DNA formation and infectious particle secretion. hNTCP­expressing PPHs were susceptible to infection with HBV particles purified from pooled HBV­positive sera, but were poisoned by raw HBV­positive sera. The use of HBV­infected hNTCP­expressing PPHs for viral entry inhibitor screening was revealed to be applicable and reproducible. In conclusion, hNTCP­expressing PPHs may be valuable tool for investigating HBV infection and antiviral drugs.

Generation of tryptophan hydroxylase 2 gene knockout pigs by CRISPR/Cas9-mediated gene targeting.

Unbalanced brain serotonin (5-HT) levels have implications in various behavioral abnormalities and neuropsychiatric disorders. The biosynthesis of neuronal 5-HT is regulated by the rate-limiting enzyme, tryptophan hydroxylase-2 (TPH2). In the present study, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system was used to target theTph2 gene in Bama mini pig fetal fibroblasts. It was found that CRISPR/Cas9 targeting efficiency could be as high as 61.5%, and the biallelic mutation efficiency reached at 38.5%. The biallelic modified colonies were used as donors for somatic cell nuclear transfer (SCNT) and 10Tph2 targeted piglets were successfully generated. These Tph2 KO piglets were viable and appeared normal at the birth. However, their central 5-HT levels were dramatically reduced, and their survival and growth rates were impaired before weaning. TheseTph2 KO pigs are valuable large-animal models for studies of 5-HT deficiency induced behavior abnomality.

[UBE2B gene and male infertility: an update].

Male infertility is a worldwide problem, and about 15% of the cases are associated with spermatogenesis-related gene mutation. The mammalian gene UBE2B is the homolog of the RAD6 gene of yeast, belonging to the ubiquitin proteasome system and playing an important role in spermatogenesis. Mice lacking the UBE2B gene are infertile, with reduced sperm motility, increased morphologically abnormal sperm, and inhibited meiosis of spermatogonia. Accumulated evidence shows that UBE2B gene mutants and single nucleotide polymorphisms are associated with male infertility. This article reviews the relation between the UBE2B gene and male infertility, offering some theoretical evidence for the diagnosis and treatment of male infertility.

Maintenance of retinal cancer stem cell-like properties through long-term serum-free culture from human retinoblastoma.

Previous studies have demonstrated that a small population of cancer stem cell-like cells exists in retinoblastoma. To provide a model for studying this population, we sought to establish a long-term culture from human retinoblastoma that have cancer stem cell-like properties. Fresh tumor tissue was digested and cultured in serum-free medium. Tumor spheres formed and were passaged continuously. Stem cell properties were examined through immunostaining, real-time quantitative RT-PCR and chemoresistance assay. Tumorigenicity of the tumor sphere-forming cells was confirmed by xenograft experiments. Furthermore, we examined the expression of cell surface markers CD44 and CD133. Tumor cells expanded as floating spheres for more than 30 passages. Sphere-forming cells overexpressed stem cell genes Oct­4, Nestin and Pax6. Immunostaining of spheres showed positivity for Nestin, Pax6 and also ABCG2. In contrast, differentiated cells derived from these spheres expressed high levels of mature retinal cell markers MAP2, GFAP, recoverin, Opsin B and Nrl, and showed immunoreactivity for NF200, GFAP, recoverin and PKCα. Furthermore, both CD44 and CD133 were highly expressed in sphere-forming cells vs. differentiated cells. Sphere-forming cells displayed higher chemoresistance to carboplatin as opposed to differentiated cells. Moreover, intraocular injection of as few as 2x103 sphere-forming cells into NOD/SCID mice gave rise to new tumors similar to the original patient tumors. These results revealed that the sphere-forming cells preserved their stem cell properties and tumorigenicity, even after long-term culture. This would be a suitable in vitro model to study cancer stem-like cells in retinoblastoma and to develop chemotherapeutic drugs and strategies.