Research progress of Tc1/Mariner superfamily.

With the rapid improvement of sequencing techniques, more and more genome annotations reveal the transposons are the important components of most genomes and present on almost all organisms. Among them, the Tc1/Mariner superfamily represents the most widespread DNA transposons. Until now, fourteen active Tc1/Mariner transposons (Minos, Mos1, etc.) have been identified and some highly active artificial transposons have been created through molecular reconstruction, such as Sleeping Beauty (SB). The transposons such as SB and Mos1 have been widely used as gene transfer vectors in the fields of transgenosis, gene trapping and gene therapy. In this review, we summarize the structure, classification, distributions, transposition mechanism and excavations of active members of Tc1/Mariner as well as its application in the fields of transgenesis, gene trapping and gene therapy.

Polymorphisms in intron 1 of the porcine POU1F1 gene.

This study was conducted to detect polymorphisms in intron 1 of porcine POU1F1 (POU domain, class 1, transcription factor 1, Pit1, renamed as POU1F1) by comparative sequencing. Within the intron, 23 sites of variation were identified, including 16 single-nucleotide substitutions, 4 single-nucleotide indels, 2 short (3-bp and 17-bp), and one long (313-bp) indels. Several important regulatory motifs were found within the 313-bp indel by in silico analysis. The 313-bp indel was next genotyped in 11 Chinese native pig breeds and 4 western meat-type pig breeds. The appearance of genotypes varied between breeds: among Chinese native breeds, no AA and AB genotypes were found in Tibetan, Lingao, Min, Rongchang, and Songliao Black pigs, no AA genotype was found in Fenjing and Leping Spotted pigs, whereas in Pietrain and Landrace there were no BB genotypes, and all 19 Duroc pigs were AA homozygotes. The western meat-type pigs had high A allele frequencies and the Chinese pigs had more B alleles, except Jianquhai pigs. A positive association of the AA genotype with birth weight was observed in a commercial pig line. This paper demonstrated that the genetic variation in intron 1 of the pig POU1F1 gene was high and these polymorphisms may provide useful makers for QTL analysis.

[Advances in studies of Sleeping Beauty transposon].

The Sleeping Beauty (SB) transposon is a Tc1/mariner family transposon that has been transpositionally inactive for over 10 million years. The SB transposon system was awakened from inactive Tc1-like transposable elements by using molecular phylogenetic data in 1997. Recent studies on its transposition efficiency and mechanism have shown its broad applications in vertebrate animals for gene-screening, gene transfer, and human gene therapy. In this review, we summarize our current knowledge of Sleeping Beauty, such as structure, transposition mechanism and potential applications, and bring forward some means aiming at the limitations of transposon technology.

Transfection and expression of exogenous gene in laying hens oviduct in vitro and in vivo.

To examine whether or not the regulatory sequence of chicken ovalbumin gene can drive transgene expression specifically in hen oviduct, the authors constructed an oviduct-specific expression vector (pOV), containing 3.0 kilobases (kb) of the 5'-flanking sequence and 3.0 kb of the 3'-flanking sequence of the chicken ovalbumin gene. Jellyfish green fluorescence protein (EGFP) reporter gene and bacterial LacZ reporter gene were respectively inserted into the downstream of the 5'-regulatory region. The recombinants were named as pOVEGFP and pOVLacZ. Two transfer systems, in vitro and in vivo, were used to verify the function of the vector. In vitro, the plasmid DNA pOVEGFP and pEGFP-N1 were transfected respectively by the polyethyleneimine procedure into the primary chicken oviduct epithelium (PCOE) and fibroblasts cells isolated from laying hens. In vivo, the recombinant vector pOVLacZ was injected into egg-laying hens via wing vein and the tissues were collected for RT-PCR analysis. The results showed that expression of pEGFP-N1 was achieved at low level in oviduct epithelial cells and at high level in fibroblasts, but that the recombinant vector was not expressed in both cells. RT-PCR analysis showed that the LacZ gene was transcribed in the oviduct, but not in the heart, liver, kidney and spleen of the injected hens. Accordingly, the beta-galactosidase activity was only detected in the oviduct magnum (116.7 mU/ml) and eggs (16.47 mU/ml). These results indicated that the cloned regulation regions of chicken ovalbumin gene could drive exogenous gene expression specifically in the oviducts of hens. In vivo gene injection via wing vein may serve as a rapid production system of recombinant proteins in chicken eggs. In addition, the cultured primary oviduct cells from laying hens were not efficient temporary expression systems for analyzing the function of regulating elements of ovalbumin gene.

[Variation and homologous analysis of parts of pig POU1F1 gene].

By PCR approach, exon4, exon5 and exon6 of POU1F1 gene of Landrace, Duroc, Yorkshire, Jiangquhai, Meishan, and w Xiangzhu are amplified, and PCR products contained exon 4,exon6 and the cloning product of exon5 were sequenced. The results showed that exon4 of POU1F1 gene had the Nla III polymorphisms characteristics, the mutation was T-->C. The sequences were digested by Nla III, and two different genotypes(GG and HH) were produced; but exon 5 and exon 6 of POU1F1 gene were high conservation in the six pig breeds. The result of homologous analysis showed that the homologous rate of nucleotide sequences of the exon4 in human, pig, mouse and cow was 93.9%,86.7%,92.1% respectively and the amino acid sequence of POU specific domain encoded by exon4 of POU1F1 gene in human, pig ,mouse and cow was identical. And the result of homologous analysis of nucleotide sequences encoding POU1F1 POU-homeo domain in human, pig, mouse, cow was 91.4%,85.1%,87.9% respectively, the homologous rate of the amino acid sequence of POU-homeo domain was 96.6%,94.8%,90.2% respectively. This showed that the sequences of the POU1F1 POU-homeo domain and the POU-specific domain encoded by exon4 in mammals were high conservation, pigs could be used to establish animal models of related diseases, providing scientific foundation and serving for human medicine.

Study on pig growth hormone gene polymorphisms in western meat-type breeds and Chinese local breeds.

Chinese Meishan and Jiangquhai pigs are two of the most prolific pigs in the world, but their growth rate is lower than that of Duroc, Landrace and Pietrain pigs. It is suggested that growth rate is regulated by growth hormone. The objective of the current study was to analyze the porcine growth hormone (pGH) gene polymorphisms based on the polymerase chain reaction restriction fragment-length polymorphism method (PCR-RFLP) for three western meat-type breeds (Duroc, Landrace and Pietrain) and two local Chinese pigs (Meishan and Jiangquhai). Five polymorphic restriction sites were detected with the ApaI, MspI, BspI and HhaI restriction enzymes in two amplified fragments (605 bp, -119 to +486; 506 bp, +206 to +711). Breed difference was found only in the 506 bp fragment. There was no difference in allelic frequencies of BspI and HhaI restriction sites among the five breeds (P>0.05). Landrace and Meishan pigs lacked allele G3 of MspI site. The allele G3 frequency of restriction MspI site of the 506 bp fragment in Pietrain pigs was higher than that in Duroc and Jianquhai pigs (P<0.001). For ApaI site, the Meishan pigs lacked allele G1; no difference was found in allelic frequencies among Pietrain, Duroc, Landrace and Jiangquhai pigs (P>0.05). This new and rapid PCR-RFLP typing method is an attractive tool for analysis of porcine growth hormone gene restriction sites. The differences in MspI and ApaI restriction sites may explain the growth difference between the foreign meat-type breeds above mentioned and local Chinese pigs.