[Design and practice of the case study methodology throughout the "Animal Genetic Principles and Breeding Methods" course].

"Animal Genetics Principles and Breeding Methods" is a main course for Master students majoring in Agriculture (Livestock) and involves a combination of theory and practice. The traditional teaching method is difficult not only to meet the requirements of modern professional degree teaching, but also for the students to master the theory and practice of genetic breeding. We have employed the case study methodology during the entire course. This paper analyzes the connotation and characteristics of the method and expounds the design and discussion of the cases. Besides, the teaching evaluation is also included. It provides a reference for the application and promotion of the case teaching method in training graduate students majoring in agriculture.

Bone marrow mesenchymal stem cell transplantation alleviates radiation-induced myocardial fibrosis through inhibition of the TGF-ß1/Smad2/3 signaling pathway in rabbit model.

Background: and purpose: Radiotherapy (RT) is an effective treatment for most malignant chest tumors. However, radiation-induced myocardial fibrosis (RIMF) is a serious side effect of RT. Currently, due to the mechanism of RIMF has not been fully elucidated, there is a lack of effective therapeutic approach. In this study, we aimed to investigate the role and possible mechanisms of bone marrow mesenchymal stem cells (BMSCs) in the therapy of RIMF. Materials and methods: Twenty-four New Zealand white rabbits were allotted into four groups (n = 6). Rabbits in the Control group received neither irradiation nor treatment. A single dose of 20 Gy heart X-irradiation was applied to the RT group, RT + PBS group and RT + BMSCs group. Rabbits in the RT + PBS group and RT + BMSCs group were injected with 200 µL PBS or 2 × 106 cells via pericardium puncture 24 h following irradiation, respectively. Echocardiography was used to test the cardiac function; Then the heart samples were collected, and processed for histopathological, Western blot and immunohistochemistry investigations. Results: It was observed that BMSCs have therapeutic effect on RIMF. Compared with the Control group, inflammatory mediators, oxidative stress and apoptosis were significantly increased, meanwhile, cardiac function was remarkably decreased in the RT group and RT + PBS group. However, in the BMSCs group, BMSCs significantly improved cardiac function, decreased inflammatory mediators, oxidative stress and apoptosis. Furthermore, BMSCs remarkably reduced the expression level of TGF-ß1 and the phosphorylated-Smad2/3. Conclusions: In conclusion, our research indicates BMSCs have the potential to alleviate RIMF through TGF-ß1/Smad2/3 and would be a new therapeutic approach for patients with myocardial fibrosis.

[Generation of goat H-FABP overexpression transgenic mice by testicular injection].

To explore the possibility of transgenic animals by testicular injection, the goat heart-type fatty acid binding protein (H-FABP) expression vector pEGFP-H-FABP was injected into the testis of 6 mice randomly by liposome mediated transfection. By detection of testis slice, sperm fluorescence and sperm DNA PCR, the exogenous gene was expressed in the parental mice. The exogenous gene was expressed at different levels in both the F1 generation mice gave birthed by treated male mice and normal female mice and the F2 generation mice generated by mating F1 could be detected that the exogenous gene expressed at different levels with the positive rates of 4% and 30.23%, respectively. The results suggested that testicu-lar injection, as an effective method to generate transgenic animal, could realize the stable integration of exogenous gene. The amelioration and maturity of testicular injection provides theoretical and practical significance in generation of trans-genic animals and even in the animal trait improvement and breeding.

Transgenic sperm produced by electrotransfection and allogeneic transplantation of chicken fetal spermatogonial stem cells.

To study self-renewal, genetic modification, and differentiation of avian spermatogonial stem cells (SSCs), we isolated chicken SSCs from fetal testes on the 16th hatching day via enzyme digestion, and then cultured the SSCs over 2 months after purification in vitro. SSCs were identified by alkaline phosphatase staining and SSEA-1 fluorescence. The EGFP gene was transfected into SSCs by three different methods: electroporation, liposome transfer and calcium acid phosphate precipitation. The transfection rate and cell survival rate using electroporation were higher than when using liposomes or calcium acid phosphate (20.52% vs. 9.75% and 5.61%; 69.86% vs. 65.00% and 51.16%, respectively). After selection with G418 for 8 days, the transgenic SSCs were transplanted into the testes of cocks treated with busulfan. Twenty-five days after transplantation, the recipients' semen was light ivory in color, and the density of spermatozoa was 3.87 (x10(7)/ml), with 4.25% expressing EGFP. By 85 days after transplantation, the number of spermatozoa increased to 32.7 (x10(7)/ml) and the rate of EGFP expression was 16.25%. Frozen sections of the recipients' testes showed that transgenic SSCs were located on the basal membrane of the seminiferous tubules and differentiated into spermatogenic cells at different stages. The EGFP gene was successfully amplified from the DNA of all recipients' semen samples.

[Analysis of genetic variations at M857 locus of the a1-Fucosy- trans-ferase (FUT1) ORF in pigs].

Enterotoxigenic Escherichia coli F18 (ETEC F18) is the main pathogen that causes edema disease and post-weaning diarrhea in piglets, and a1-fucosytransferase (FUT1) gene has been identified as a receptor gene encoding the receptor for ETEC F18 bacteria. In this study, the method of PCR-RFLP was used to investigate the among 21 breeds including one wild boar breed and 20 western commercial and Chinese native pig breeds (populations). The results showed that none of the individuals in all 21 breeds possessed the resistant AA genotype, the genetic polymorphisms of the FUT1 locus were only detected in two western pig breeds (Duroc and Yorkshire), Lingao pig and hybrid pig breeds, while the wild boar and all the other Chinese pig breeds only possessed the susceptible GG genotype. The results indicated that Chinese native pig breeds, unlike western pig breeds, lack the genetic background on the resistance to ETEC F18 bacteria. This may be owe to their different origination, as the resistance gene to ETEC F18 might be originated from European wild boar. It was also inferred that edema disease and post-weaning diarrhea caused by ETEC F18 had close relationship with the growth speed of pigs.

[Analysis of polymorphisms in exon 14 in porcine Mx1 gene and identification of new mutation points].

By the PCR-RFLP method, the polymorphisms of exon 14 of Mx1 gene were detected in 7 native and foreign pig breeds. Taken together with the analysis of restriction enzyme Hin6 I digestion, there were 6 genotypes and 3 alleles. And, all individuals of Duroc exhibited the AA genotype but the Sutai pigs exhibited all three kinds of genotypes. The BB genotype was detected only in Meishan pigs and their derivate Sutai pigs. Among all pig breeds, the allele B only appeared in Chinese indigenous pig breeds and developed pig breed in this study, and the allele A was dominant allele in all pig breeds except for Songliao black pig. The results of Chi Square test showed that there were abundant polymorphisms in all pig breeds. The gene frequency of Meishan pig and Songliao black pig showed greatly significant difference to other pig breeds (P<0.01), Sutai pig showed greatly significant difference (P<0.01) to other pig breeds except for Pietrain; and Huai pig showed greatly significant differences (P<0.01) with Pietrain and other indigenous Chinese pig breeds, but no difference with Duroc and Yorkshire (P>0.05). Three new mutation points in three genotypes were identified by sequencing comparison analysis of PCR products, two of which resulted in Thr-->Ala and Glu-->Arg respectively, the last one was a silence mutation, the Glu-->Arg mutation point and this silence mutation point only appeared in the individuals with the BB genotype.

[Studies on taihe silkies' karyotype and band pattern].

This study made the chromosome slides of Taihe Silkies by the peripheral blood lymphocyte culture-drying method,and analyzed Taihe Silkies karyotype and band pattern. The results are as follow: The diploid chromosome number of Taihe Silkies was 2n=78,the basic number of chromosome arms was AF=90 and the sex chromosome type was ZZ(male symbol)/ZW(female symbol). According to the measured relative length,arm ratio and centromeric index,the first 10 pairs of macro-chromosomes are described as follows:No.1 ,9 and Z,W chromosomes were metacentrics,No.2,4,7 were submetacentrics,and No.3,6,8,10 were telocentrics. Studies on Taihe Silkies' G-band showed that the first 10 pairs of macro-chromosomes can be divided into 29 zones and 190 bands. Being treated by C-banding technique,a totally dark-stained and easily indentified W-chromosome always showed up in the female metaphase configurations. Ag-NORs were located in the short arms' telomere of No.1,2 euchromosomes and Z sexchromosome,the Ag-NORs number varied from 1-6. -NORs.

[Studies of chromosome Ag-NORs of Xianju chickens and the relation with the productive performance].

By using the method of culturing peripheral blood, Ag-NORs patterns of Xianju chickens were investigated. The results are as follows:Xianju chickens' Ag-NORs were located in 1p,3p,4p,Zp and 2q. The mean value per cell was 3.26 and the mode was 3. The size, the number and distribution between different individuals or of the same individual were polymorphic,and associations of Ag-NORs were found. Performances between two groups with different Ag-NOR frequency were not significantly different(P>0.05) except shank length in Xianju cocks.

[Karyotypes and G-banding patterns of quail].

Karyotype analysis was carried out on quails using the peripheral lymphocyte culture techniques,and G-banding patterns were obtained with trypsin and Giemsa. The results showed that the quail had a diploid number of 78, with 10 pairs of macrochromosomes including the sex-chromosomes and 29 pairs of microchromosomes. NO.1 chromosome was submetacentric, NO.2 and Z chromosome were metacentric,and the others were telocentric, which was slightly different from the results of others. Additionally,analysis of G-banding patterns for macrochromosomes indicated that they were divided into 27 zones and 134 bands.

A screen of suitable inducers for germline differentiation of chicken embryonic stem cells.

Differentiation of germ cells from embryonic stem cells in vitro could have great application for treating infertility and provide an excellent model for uncovering molecular mechanisms of germline generation. In this study, we aim to screen the suitable inducers that may prove the efficiency of driving chicken embryonic stem cells (ES cells) toward germ cells. The male ES cells were separeted into different groups: single retinoic acid (RA) treatment, co-cultured with sertoli cell feeder with RA induction, cultured on matrix proteins (fibronectin, laminin and collagen) with RA treatment, cultured on fibronectin with sertoli cell feeder and RA induction, and single bone morphogenetic protein 4 (BMP4) treatment. Quantitative RT-PCR and immunoourescence were performed to characterize the ES cells differentiation process. The results showed that spermatogonial stem cells (SSCs)-like were not detected in single RA and RA with collagen groups, but were observed in the other groups. The expression of ES specific genes (Nanog and Sox2) was decreased while SSCs marker genes (Dazl, Stra8, integrin α6, integrinß1 and C-kit) was remarkably increased. The multiple comparsion results showed that the expression of SSCs marker genes in RA with sertoli cells group was significantly higher than the other groups(P<0.05). Collectively, our results suggested that chicken ES cells possess the potency to differentiate into SSCs-like cells in vitro through RA, matrix proteins, sertoli cells and BMP4 induction, of which co-cultured with sertoli cell feeder with RA induction was proved to be the best.

Xuhuai goat H-FABP gene clone, subcellular localization of expression products and the preparation of transgenic mice.

The aim of this study was to clone the heart-type fatty acid binding protein (H-FABP) gene of Xuhuai goat, to explore it bioinformatically, and analyze the subcellular localization using enhanced green fluorescent protein (EGFP). The results showed that the coding sequence (CDS) length of Xuhuai goat H-FABP gene was 402 bp, encoding 133 amino acids (GenBank accession number AY466498.1). The H-FABP cDNA coding sequence was compared with the corresponding region of human, chicken, brown rat, cow, wild boar, donkey, and zebrafish. The similarity were 89%, 76%, 85%, 84%, 93%, 91%, 70%, respectively. For the corresponding amino acid sequences, the similarity were 90%, 79%, 88%, 97%, 95%, 94%, 72%, respectively. This study did not find the signal peptide region in the H-FABP protein; it revealed that H-FABP protein might be a nonsecreted protein. H-FABP expression was detected in vitro by reverse transcription-polymerase chain reaction (RT-PCR), and the EGFP-H-FABP fusion protein was localized to the cytoplasm. The gene could also be transiently and permanently expressed in mice.

[Establishment of a transgenic cell model for preliminary screening of chemopreventive agents].

To develop a GFP transgenic cell model under the transcriptional control of TK promoter adjacent to which ARE enhancer was inserted. Synthetic oligonucleotide ARE motif was annealed and purified then inserted into pTK-GFP to construct the vector of pARE-TK-GFP. The TK and ARE-TK fragments were amplified by PCR and cloned into pEGFP-N1 to reconstruct eukaryotic expression vectors of pTK-GFP/Neo and pARE-TK-GFP/Neo. They were transfected into HepG2 cells and clones resistant G418 were isolated. PDTC and Lentinan were used to induce the cell levels of GFP and the fluorescence was measured using a fluorescence plate reader. The results showed that the induced level of GFP is significantly increased and have dose-dependeny in a certain range. This findings indicated that such a cell model offered a potential platform for preliminary screening of all kinds of natural or synthetic chemopreventive agents.

[Molecular genetic diversity of Fujian domestic duck breeds].

By using 28 micro-satellite markers with better polymorphism, this paper studied the genetic diversity of four Fujian provincial domestic duck breeds Jinding, Putian black, Liancheng white, and Shanma. According to the alleles frequencies, the polymorphic information content, average heterozygosity, anaqular genetic distance (DA) and Nei' s standard genetic distance (DS) for each breed were calculated. Based on DA and DS, four dendrograms were obtained by neighbor-joining (NJ) and UPGMA methods. The results showed that the average heterozygosity of the four duck breeds was 0. 5353, indicating that the protection of the genetic diversity of these breeds should be strengthened. The orders of the two types of genetic distances among the breeds were accordant, and the dendrograms were the same, reflecting that much more micro-satellite loci should be adopted to obtain more universal conclusions when the genetic diversity was analyzed. The phylogenetic relationships among the four duck breeds were in accordance with their economic types and ecological localities.