Application of genetic modification technologies in molecular design breeding of sheep.

Genetic modification technologies can be used for modifying animal genome to express exogenous genes or affect the function of endogenous genes. In animal breeding, genetic modification technologies allow the rapid generation of germplasms with beneficial traits. It includes traditional genetic modification, virus or sperm carrier-mediated genetic modification and nuclease-mediated genome editing, especially the CRISPR/Cas9, one of the artificial nuclease genome editing technologies, have been applied in genome editing in many domestic animals including sheep (Ovis aries). Compared with conventional strategies used for animal breeding, there is great value for sheep breeding improvement by using genome editing technology, which is more effective and timesaving. In this review, we summarize the approaches of genetic modification in sheep and discuss the possibility of molecular design and breeding of sheep by genome editing technologies. We also identify the potential bottlenecks and challenges of these technologies in sheep breeding.

[Screening of tissues pooled cDNA library using probes by restricted fragments of BAC positive clones of ovine MHC].

Under the premise what we have known bacterial artificial chromosome(BAC)clone sequence information and gene annotation predicted in the Chinese Merino sheep major histocompatibility complex (MHC) region, the digested fragments from 6 BAC clones that were located in the MHC region of the Chinese Merino sheep genome BAC library, which were used to screen the cDNA library using plaque in situ hybridization as probes. The full length of positive cDNA clones (sequences) isolated were completely sequenced, and the sequences obtained were aligned with the corresponding known sequence information and the BAC clones with gene annotation. Meanwhile, the sequence similarity was searched in NCBI Blastn database. This work aimed at verification of accuracy of the gene annotation results and initial analysis of gene (sequence) function. At last, 27 positive cDNA clones (sequences) in total were screened through two runs of hybridization. It was also found that these sequences could be positioned in the corresponding BAC clones, and 25 sequences were located in exon area of the annotated gene. It was verified that 23 sequences had the highest sequence similarity with those in the Bos taurus by searching against the NCBI Blastn database; moreover, the function of these sequences were closely relate to immunology.

[Confirmation and expression analysis of three predicted genes in sheep MHC region].

Previous DNA sequencing of BAC clones covering entire ovine MHC (OLA) region resulted in identification of approximately 130 functional genes in the region, of which 8 were predicted by computer software to be exclusively existed in sheep, but not in any other species known to date. In the present study, we successfully identified and cloned cDNA sequence of OaN2, OaN5, and OaN6 from representative sheep tissues, confirmed their existence in reality. The sequences obtained experimentally exactly identical to those predicted previously. The length of cDNA fragments for OaN2, OaN5, and OaN6 was 270 bp, 309 bp, and 205 bp, respectively, with GenBank accession number assigned as JF330782 (OaN2), JF330783 (OaN5), and JF330784 (OaN6). Northern analyses indicated that the mRNA transcripts of OaN2 were mainly seen in ovine mesenteric lymph nodes and spleen, while OaN5 was observed in only in mesenteric lymph nodes. In contrast, OaN6 transcripts were detected in all tissues except for liver and heart. Western blot showed that OaN2 protein expression level was detected in mesenteric lymph nodes, spleen, and liver, essentially consistent with that of mRNA transcripts. Immunohistochemistry analysis showed that OaN2 protein was highly expressed in ovine mesenteric lymph nodes, moderately expressed in, and not expressed in heart, liver, and pancreas, consistent with the results of Western blotting. The cloning and expression analysis of 3 novel genes provide a basis for revealing their specificities and would be helpful to further study of their expression profile and their potential functions.

[Cloning, expression analysis and RNA interference of FGF5 gene in sheep].

The cDNA of fibroblast growth factor 5 (FGF5) gene in sheep was cloned, and the nucleotides sequence homology of FGF5 was compared with other six mammal. In addition, the expression of FGF5 in different tissues was analysed. Gene FGF5 was then recombined into prokaryotic expression vector (pGEX-4T-2) and RNA interference vector (pSilencer 5.1 H1) to study its expression in fibroblast cell lines. Results showed that the open reading frame (ORF) of cDNA in sheep consisted of 813 nucleotide acids encoding 270 amino acids, with the molecular mass of 29.58 kDa and theoretical pI of 10.59. The amino acids sequence of FGF5 gene in sheep shared high identity with those in cow, human, mouse, rat, dog, cat and rabbit. In addition, analysis on tissue expression showed that FGF5 expressed in skin, heart, kidney, liver, pancreas, spleen, lung, and small intestine, especially presenting high levels in skin. The expression of FGF5 in E. coli was induced with IPTG, which produced a protein band with the expected size of 56 kDa on SDS-PAGE, while the expression of FGF5 in sheep fibroblast cell line was knocked down remarkably with the help of integrated RNAi vector.

[Production of porcine blastocysts expressed EGFP by handmade cloning].

Production of transgenic animals via somatic cell nuclear transfer (SCNT) has been widely used worldwide. However, the application of SCNT is impeded by overall high costs and low efficiency. Here, we reported a modification of the existing technology in order to overcome some of the disadvantages associated with SCNT. Firstly, a marker gene, enhanced green fluorescent gene (EGFP), was transfected into pig fetal fibroblast cells, and was subsequently screened by fluorescent expression to ensure donor cells expressing EGFP. Porcine embryos expressing EGFP were then produced by a method called handmade cloning (HMC), a simplified method for micromanipulation. To demonstrate the concept, we collected a total of 378 fresh swine oocytes, from which 266 with the nucleus removed, obtained a total of 127 viable recombinant oocytes after fusion with EGFP-expressing cells. In vitro incubation of the 127 recombinant oocytes for approximately 144 hours resulted in successful generation of 65 viable embryos, with an average success rate of 52.1±8.3%. Compared with the traditional SCNT, the method of HMC is not only easy to operate, but also increases the rate of recombinant embryo significantly. Furthermore, the modified method no longer relies on expensive instrument like micromanipulator, facilitating the industrialization of transgenic animal production.

The ADH1B Arg47His polymorphism in east Asian populations and expansion of rice domestication in history.

BACKGROUND: The emergence of agriculture about 10,000 years ago marks a dramatic change in human evolutionary history. The diet shift in agriculture societies might have a great impact on the genetic makeup of Neolithic human populations. The regionally restricted enrichment of the class I alcohol dehydrogenase sequence polymorphism (ADH1BArg47His) in southern China and the adjacent areas suggests Darwinian positive selection on this genetic locus during Neolithic time though the driving force is yet to be disclosed. RESULTS: We studied a total of 38 populations (2,275 individuals) including Han Chinese, Tibetan and other ethnic populations across China. The geographic distribution of the ADH1B*47His allele in these populations indicates a clear east-to-west cline, and it is dominant in south-eastern populations but rare in Tibetan populations. The molecular dating suggests that the emergence of the ADH1B*47His allele occurred about 10,000 to approximately 7,000 years ago. CONCLUSION: We present genetic evidence of selection on the ADH1BArg47His polymorphism caused by the emergence and expansion of rice domestication in East Asia. The geographic distribution of the ADH1B*47His allele in East Asia is consistent with the unearthed culture relic sites of rice domestication in China. The estimated origin time of ADH1B*47His allele in those populations coincides with the time of origin and expansion of Neolithic agriculture in southern China.

Winter temperature and UV are tightly linked to genetic changes in the p53 tumor suppressor pathway in Eastern Asia.

The tumor suppressor p53 is a master sensor of stress. Two human-specific polymorphisms, p53 codon 72 and MDM2 SNP309, influence the activities of p53. There is a tight association between cold winter temperature and p53 Arg72 and between low UV intensity and MDM2 SNP309 G/G in a cohort of 4029 individuals across Eastern Asia that suggests causative selection. Moreover, the two polymorphisms are not coselected. Haplotype-based selection analysis further suggests that this is a striking example of two functional polymorphisms being strongly selected for in human populations in response to environmental stresses.

[Construction and analysis of rumen bacterial artificial chromosome library from a dairy cow rumen microflora].

The high molecular weight DNA was extracted and purified directly from rumen samples in the study by using culture-independent and pulsed field gel electrophoresis approaches. After digestion with Hind III, DNA fragments ranging from 50-100 kb was collected and ligated to pCC BAC vector. The ligation mixture was transformed into E. coli EPI300 and a rumen metagenomic BAC library with about 15360 clones was constructed. The average insert size is about 54.5 kb, mostly ranging from 50-70 kb, and the capacity of this BAC library is about 837Mb. Several BAC clones with activity of amylase, Cmcellulase had been screened from the BAC library. The clones with Cmcelluase activity were screened further for linchenase, xylanase, cellobioase activity and the result is that 25 of them have at least one kind of other enzyme activity.

[Constrcution and application of Xinjiang fine-fleece sheep 2 times genome BAC library PCR screening system].

In this study, we used our special regional fostering breed Xinjiang fine-fleece sheep's genome to construct its genome library. The average size of insert fragments of the library was 133 kb. At the same time 92.5% clones' insert fragments of the library were larger than 100 kb and some larger than 300 kb. If we supposed sheep's genome contenting 3 x 10(6) kilo-base, on the basis of the average insert fragment was 133 kb the library covered 8 times genome of Xinjiang fine-fleece sheep. The probability of the tagged fragment being screened from the library was 98.208%. To prove the BAC library had been the better rate of coverage,four molecular markers: DMB_EX2, MCMA36, CP73 and BM1258 located to MHC gene of chromosome 20 near region in xinjiang fine-fleece sheep had been screened positive clones from the constructed 2 times genome library PCR screening system and the average positive clones was 1.5. The screening result showed that the constructed genome library was fairly closed to the 8 times genome coverage and had no erroneous tendency, which made the library being of the utmost useful resource for studying functional gene, position cloning and improving the genome physical map of sheep.

[Molecular detection of specific HPV types in Condylomata acuminata].

To detect HPV in genital warts (Condylomata acuminata, CA) for infection rate and association of specific HPV types between males and females, and to provide support for the development of HPV vaccines, we designed HPV type-specific oligonucleotide primers to amplify DNA fragments encoding L1 viral capsule protein. SSP-PCR was conducted in duplication for each CA sample from male and female patients. DNA of TA-cloned HPV was used as positive control, and deionized H2O was used as negative control. A total of 22 clinical samples, 13 from males and 9 from females, was collected from patients diagnosed with CA at hospitals in Beijing and Handan. HPV viral DNA was amplified in all 22 samples analyzed, with 100% detection rate. TA-cloning and sequencing of the PCR products confirmed correct amplification of HPV type-specific fragments. Of the 13 samples from males, 5 were infected with HPV6, 6 with HPV11, and 2 with HPV6 + HPV11. Of the 9 samples from females, 3 were infected with HPV6, 2 with HPV11, and 4 with both HPV6 and HPV11 infection. In addition, high-risk types HPV16, HPV18, HPV33, HPV35, HPV45, HPV54, HPV56 and HPV58 were also detected in 4 female samples that were mixed with cervical cell debris during sample collection. However, no HPV types other than HPV6 and HPV11was detected in all CA-only samples in this study. We have established a sensitive and reliable laboratory procedure for HPV detection and classification. Using the method, we reached 100% detection rate of HPV in the CA samples. Our results confirm that HPV6 and HPV11 are primarily responsible for CA, and there is no specific association of HPV types between warts in males and females.

[Molecular Cloning of Smooth Muscle Calponin h1 in SHeep.].

It was found that the level of Calponin h1 (CaP h1) mRNA was significantly up-regulated by Estrogen in the myometrium of sheep towards the end pregnancy. Although the CaP h1 has been widely used as a reference gene to observe the changes of expression level of other genes, the full-length gene in sheep has not been obtained. With the oligo nucleotide primers according to human, mouse and pig CaP h1 mRNA, the full-length cDNA of CaP h1 was cloned by 5'- and 3'-RACE (Genbank accession number = AY327118). The cDNA was 1499bp in length and contained a complete open reading frame of 891 bp, encoding a protein of 297 amino acid residues. 5'-and 3'-UTR was 79 bp and 529bp, respectively. With PCR-SSP approaches,the genomic DNA of sheep CaP h1 was obtained .It showed that the gene has 7 exons and 6 introns, spanning over 8kb(Genbank accession number of introns : AY771807,AY771808, AY771809, AY771810.) Homologous comparison indicated that the cDNA sequences are highly conserved across the species. The highest homology was found in wild pig (92%), followed by human (88%), rat (81%), mouse (81%) and chicken (79%). The intron sequence and length showed a large variation among species (>50%).

[Identification and characterization of alternativly splicing variants for murine mater gene].

Mater encoding an oocyte-specific autoantigen,and is associated with premature autoimmune ovarian dysgenesis (AOD) in mouse. Based on RT-PCR, cDNA cloning, screening, sequencing and analysis, we have detected a total of four Mater splice variants, designated as variant B, E, F and G. All these splicing forms are in frame in terms of expected protein products. Among these, B was consistent with the previous report, whereas E, F, G belong to novel splice variants that have not been reported previously. Variant E lacks exon 6, variant F both lacks exon 10 and retains a part of intron 8, variant G lacks part of exon 14, and variant H lacks part of exon 13. The cDNA sequences at all the exon-intron boundaries confirms to the "GT-AG" splicing rule. Variant B, E, F exist in all the four strains. Variant G exists only in SWR/J. According to the cDNA sequences of these four splice variants, amimo acid sequences of the corresponding expected protein isoforms were deduced, and their potential functional effects were predicted in this thesis. Further identification and characterization of these expected protein isoforms would provide valuable information for their functional importance.

[Divergence and systematical evolution of three Leuciscus species in Xinjiang based on mitochondrial DNA control region sequences].

Nucleotide sequence of fish mitochondria DNA (mtDNA) D-loop region from Leuciscus leuciscus baicalensis, L. merzbacheri, and L. idus in Xinjiang, China, were examined by sequencing 667 - 669 bp length of homological fragments in the D-loop from 24 individuals of the three fish species. DNA divergence ranged from 6.39% - 9.89% among the three fish species in the genius of Leuciscus cuvier. The sequence similarity is high and the variation is low between L. Leuciscus baicalensis and L. idus. In contract, the genetic distance is larger between L. Leuciscus baicalensis and L. merzbacheri. The average nucleotide variation within each of the two geographical populations (Sailimu lake and E' erqis river) of L. leuciscus baicalensis is 1.07% and 1.08%, respectively, and such variation is 1.07% between the two populations. These results demonstrate that the two geographic populations of L. leuciscus baicalensis do not appear to have significant genetic differentiation. Sequencing data showed the existence of sufficient genetic variations among three species of fish, as illustrated by distinct haplotypes for each species. The phylogenetic trees built with MEGA1.02 pointed out that the relationship between L. leuciscus baicalensis and L. idus is close, and L. merzbacheri is ancient among three Leuciscus.

Identification of alternate splice variants for murine SmX5.

We report here the identification of three additional murine SmX5 alternative transcripts, designated SmX5a, b and c. The cDNA of SmX5a possesses intron 1 in its entirety, and SmX5b harbors a part of intron 2. The retained fragment of SmX5c starts at the same position as SmX5b, but the retention extends through the remaining portion of intron 2. The sequence of retained DNA at all the intron-exon boundaries conforms to the "GT-AG" rule. RT-PCR analysis indicates that the three isoforms are stable transcripts, all at low frequency when compared with SmX5. Expression analysis by RT-PCR indicates that SmX5 and its isoforms exist in murine brain, kidney, testis, thymus, liver, spleen and heart. The SmX5a isoform is predominantly expressed in the thymus, while SmX5b and c are expressed in all tissues examined. The presence of alternatively spliced SmX5 isoforms and tissue-specific variation in splicing patterns suggests that SmX5 expression is complex and that the regulation of all four SmX5 mRNA levels may be critical in maintaining proper pre-mRNA splicing machinery in different cell types.

Expression of an antitumor-analgesic peptide from the venom of Chinese scorpion Buthus martensii karsch in Escherichia coli.

The gene encoding a putative mature antitumor-analgesic peptide (AGAP) from the venom of the Chinese scorpion Buthus martensii Karsch was obtained by polymerase chain reaction (PCR) according to its cDNA sequence and expressed in Escherichia coli. While most of the recombinant AGAP was expressed in the form of insoluble inclusion body. The recombinant AGAP was purified to homogeneity by metal chelating affinity chromatography. Pharmaceutical tests showed that the recombinant AGAP has both analgesic and antitumor activities on mice.