The piRNAs present in the developing testes of Chinese indigenous Xiang pigs.

The piRNA pathway plays an essential role in defense against transposable elements in the germline tissues of animals and contributes to post-transcriptional regulation of genes. Xiang pigs present an earlier sexual maturation compared with most European pig breeds, but the role that the piRNA pathway plays in the development of Xiang pigs is currently not understood. In this study, we sequenced and analyzed piRNAs expressed in the testes of Xiang pigs at four different ages, and identified endogenous piRNAs which were highly abundant at each time point. The lengths of the identified piRNAs ranged from 24 to 34 nucleotides (nt), with the most abundant length being 29 nt. Additionally, there was a strong bias for uracil at the first position, a slight bias for adenine at position 10 and frequent 5'-10 nt complementary sequences, suggesting that ping-pong-mediated silencing is present in the Xiang pig germline. We observed that the piRNA composition changed from TE-associated piRNAs in two- and three-month-old testes to predominantly gene-derived and intergenic piRNAs in six- and twelve-month-old testes, with a gradual increase in the expression level of piRNAs over the course of testis development. And more than half of piRNA reads mapped to just a few of 473 predicted piRNA clusters. Additionally, we found that several genes were highly enriched by piRNA reads, including CYP19A1, PRMT8, SUZ12, WWOX, SGSM1 and MIF. The functions of these genes are primarily associated with steroidogenesis and histone modification. Changes in piRNA composition and widespread expression patterns during spermatid development indicate that these small ncRNAs may be responsible not only for transposon suppression but also for post-transcriptional regulation of several protein-coding genes essential for normal spermatogenesis.

Identification and profile of microRNAs in Xiang pig testes in four different ages detected by Solexa sequencing.

To further understand the role of microRNA (miRNA) during testicular development, we constructed four small RNA libraries from the testes of the Chinese indigenous Xiang pig at four different ages, which were sequenced using high-throughput Solexa deep sequencing methods. It yielded over 23 million high-quality reads and 1,342,579 unique sequences. At two and three months of age, the proportion which represented miRNAs was the most abundant class of small RNAs, but it was gradually replaced by the category that represented piRNAs in adult testes. We identified 543 known and homologous conserved porcine miRNAs and 49 potential novel miRNAs. There were 306 known miRNAs which were co-expressed in four libraries. Six miRNAs and three potential novel miRNAs were validated in testes and sperms of Xiang pig by RT-qPCR method. Many clusters of mature miRNA variants were observed, in which let-7 family was the most abundant one. After comparison among libraries, 204 miRNAs were identified as being differentially expressed and likely involved in the development and spermatogenesis of pig testes. This work presented a general genome-wide expression profile of the testes-expressed small RNAs in different ages of pig testes. Our results suggested that miRNAs performed a role in the regulation of mRNAs in puberty pig testes while piRNAs likely functioned mainly in sexually mature pig testes.

A stringent dual control system overseeing transcription and activity of the Cre recombinase for the liver-specific conditional gene knock-out mouse model.

Liver cancer is one of the most threatening diseases in Chinese population. Just like in other tissues, tumor initiation and development in liver involve multiple steps of genetic and epigenetic alterations with several unknown details. However, unlike in other tissues, a tissue specific inducible Cre recombinase system that allows temporal and spatial deletion of a target DNA fragment is still not available for in vivo functional gene annotation in hepatocytes. In our pursuit to establish such a mouse model, we designed a dual inducible Cre transgene system and tested it in cultured cells. By combining a CCAAT/enhancer binding protein beta (C/EBP beta) promoter derived Tet-off expression system and the estrogen receptor (ER) mediated functional control, we show a desirable profile of both hepatocyte-specificity and regulability of the Cre expression in a series of critical assessments in the cell culture system, which provides confidence in continuation of our ongoing pursuit in mouse.

Experimental cancer gene therapy by multiple anti-survivin hammerhead ribozymes.

To improve the efficacy of gene therapy for cancer, we designed four hammerhead ribozyme adenoviruses (R1 to R4) targeting the exposed regions of survivin mRNA. In addition to the in vitro characterization, which included a determination of the sequence specificity of cleavage by primer extension, assays for cell proliferation and for in vivo tumor growth were used to score for ribozyme efficiency. The resulting suppression of survivin expression induced mitotic catastrophe and cell death via the caspase-3-dependent pathway. Importantly, administration of the ribozyme adenoviruses inhibited tumor growth in a hepatocellular carcinoma xenograft mouse model. Co-expression of R1, R3 and R4 ribozymes synergistically suppressed survivin and, as this combination targets all major forms of the survivin transcripts, produced the most potent anti-cancer effects. The adenoviruses carrying the multiple hammerhead ribozymes described in this report offered a robust gene therapy strategy against cancer.