Astragalus additive in feed improved serum immune function, rumen fermentation and the microbiota structure of early-weaned lambs.

AIM: The purpose of this study was to determine the mechanism of Astragalus activity on the immune function, rumen microbiota structure, and rumen fermentation of early-weaned lambs. METHODS AND RESULTS: Thirty healthy early-weaned lambs with similar body weights (17.42 ± 2.02 kg) were selected for the feeding experiment. The control group (KB) was fed a basal diet, and the Astragalus group (HQ) was fed 0.3% Astragalus additive on the basis of a basic diet. The formal trial period was 60 days. The results showed that the concentrations of blood immunoglobulin A (IgA) and immunoglobulin M (IgM) in the HQ group were significantly higher than those in the KB group (P < 0.05). Compared with the KB group, the concentrations of acetic acid, butyric acid, and total volatile fatty acids (VFAs) in the HQ group were higher (P < 0.01). The expression levels of the rumen epithelial-related genes MCT1, MCT4, NHE2, and ZO1 in the Astragalus group were significantly higher than those in the KB group (P < 0.05). 16S rRNA analysis showed that at the phylum level, Bacteroidetes in the HQ group significantly increased (P < 0.01); at the genus level, Prevotella (P < 0.01) and Succiniclasticum (P < 0.01) in the HQ group were found at significantly higher abundances than those in the KB group, and the results of microbiota gene and function prediction showed that "energy metabolism," "glycan biosynthesis and metabolic" pathways were significantly enriched in the HQ group (P < 0.05). CONCLUSION: As a feed additive, Astragalus can improve the immunity of early-weaned lambs, the structure of the rumen microbiota of lambs, and the fermentation capacity of the rumen.

Smad4 mediates Bmf involvement in sheep granulosa cell apoptosis.

Bcl-2-modifying factor (Bmf) functions to mediate follicular atresia and oocyte growth in mice. It has been proven that TGF-ß can induce Bmf expression via the Smad4 pathway in a variety of cells, and then induce cell apoptosis. Based on this, we hypothesized that Smad4 and Bmf may play important roles in the apoptosis of granulosa cells (GCs) in domestic animals. This study used small-tailed Han sheep follicular GCs cultured in vitro as a model system, and overexpression or interference experiments, to explore the biological roles of Bmf and reveal the preliminary regulatory mechanisms between Smad4 and Bmf in the process of GCs' apoptosis. We found that the proliferation rate of sheep GCs was significantly increased after the knockdown of Bmf, whereas overexpressing Bmf increased the apoptosis rate of GCs, results also verified by the expression patterns of PCNA, Bcl-2, and Bax genes. After the Smad4 knockdown, the apoptosis rate of GCs was increased, while the mRNA and protein expression of Bmf was significantly up-regulated. A rescue experiment verified that the Bmf knockdown could alleviate GCs' apoptosis induced by Smad4 knockdown. In conclusion, our study not only elucidated an important role for Bmf in the apoptosis of sheep GCs but also revealed a new regulatory pathway between Smad4 and Bmf in this process.

Isobaric Tags for Relative and Absolute Quantification-Based Proteomics Reveals Candidate Proteins of Fat Deposition in Chinese Indigenous Sheep With Morphologically Different Tails.

Background: Chinese indigenous sheep can be classified into two types according to their tail morphology: fat-rumped and thin-tailed sheep, of which the typical breeds are Altay sheep and Tibetan sheep, respectively. Methods: To identify the differentially expressed proteins (DEPs) underlying the phenotypic differences between tail types, we used isobaric tags for relative and absolute quantification (iTRAQ) combined with multi-dimensional liquid chromatography tandem-mass spectrometry (LC-MS/MS) technology to detect candidate proteins. We then subjected these to a database search and identified the DEPs. Finally, bioinformatics technology was used to carry out Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Results: A total of 3,248 proteins were identified, of which 44 were up-regulated and 40 were down-regulated DEPs. Analyzing their GO function terms and KEGG pathways revealed that the functions of these DEPs are mainly binding, catalytic activity, structural molecule activity, molecular function regulator, and transporter activity. Among the genes encoding the DEPs, APOA2, GALK1, ADIPOQ, and NDUFS4 are associated with fat formation and metabolism. Conclusion: The APOA2, GALK1, ADIPOQ, and NDUFS4 genes may be involved in the deposition of fat in the tail of sheep. This study provides a scientific basis for the breeding of thin-tailed sheep.

Genome wide association study for the identification of genes associated with tail fat deposition in Chinese sheep breeds.

Chinese indigenous sheep can be classified into three types based on tail morphology: fat-tailed, fat-rumped, and thin-tailed sheep, of which the typical breeds are large-tailed Han sheep, Altay sheep, and Tibetan sheep, respectively. To unravel the molecular genetic basis underlying the phenotypic differences among Chinese indigenous sheep with these three different tail types, we used ovine high-density 600K single nucleotide polymorphism (SNP) arrays to detect genome-wide associations, and performed general linear model analysis to identify candidate genes, using genotyping technology to validate the candidate genes. Tail type is an important economic trait in sheep. However, the candidate genes associated with tail type are not known. The objective of this study was to identify SNP markers, genes, and chromosomal regions related to tail traits. We performed a genome-wide association study (GWAS) using data from 40 large-tailed Han sheep, 40 Altay sheep (cases) and 40 Tibetan sheep (controls). A total of 31 significant (P<0.05) SNPs associated with tail-type traits were detected. For significant SNPs' loci, we determined their physical location and performed a screening of candidate genes within each region. By combining information from previously reported and annotated biological functional genes, we identified SPAG17, Tbx15, VRTN, NPC2, BMP2 and PDGFD as the most promising candidate genes for tail-type traits. Based on the above identified candidate genes for tail-type traits, BMP2 and PDGFD genes were selected to investigate the relationship between SNPs within the tails in the Altay and Tibetan populations. rs119 T>C in exon1 of the BMP2 gene and one SNP in exon4 (rs69 C>A) of the PDGFD gene were detected. rs119 was of the TT genotype in Altay sheep, while it was of the CC genotype in Tibetan sheep. On rs69 of the PDGFD gene, Altay sheep presented with the CC genotype; however, Tibetan sheep presented with the AA genotype.

Study on the correlation between BMPR1B protein in sheep blood and reproductive performance.

Sheep reproductive performance is one of the important economic traits in sheep farming. The bone morphogenetic protein receptor 1B (BMPR1B) gene and protein may play an important role in sheep fertility. This study was to investigate the association of blood BMPR1B protein expression with reproductive performance in sheep. Mongolian sheep with single and twin births and polytocous Small Tail Han sheep were selected due to differences in birth numbers. The BMPR1B mRNA in sheep blood was measured by a reverse transcription-polymerase chain reaction as well as the BMPR1B protein was measured by enzyme-linked immunosorbent assay in blood samples of Mongolian and Small Tail Han sheep. The results demonstrated that blood BMPR1B concentration in Mongolian sheep with twin birth was higher (P < 0.05) than Small Tail Han sheep and Mongolian sheep with single birth. The protein concentration in the anestrus season was higher (P < 0.045) than those in the estrus season for both Mongolian and Small Tail Han sheep. Moreover, BMPR1B concentration in Mongolian sheep increased (P < 0.05) at the age of 6 to 12 mo and that in Small Tail Han sheep increased (P < 0.05) at the age of 3 to 6 mo. The result indicates that the increase in BMPR1B protein concentrations in the blood of Mongolian ewes and Small Tail Han ewes may be beneficial to follicular development, but too high or too low of this blood protein concentration in Mongolian and Small Tail Han sheep is not conducive to ovulation.

Detection of copy number variation and selection signatures on the X chromosome in Chinese indigenous sheep with different types of tail.

OBJECTIVE: Chinese indigenous sheep breeds can be classified into the following three categories by their tail morphology: fat-tailed, fat-rumped and thin-tailed sheep. The typical sheep breeds corresponding to fat-tailed, fat-rumped, and thin-tailed sheep are large-tailed Han, Altay, and Tibetan sheep, respectively. Detection of copy number variation (CNV) and selection signatures provides information on the genetic mechanisms underlying the phenotypic differences of the different sheep types. METHODS: In this study, PennCNV software and F-statistics (FST) were implemented to detect CNV and selection signatures, respectively, on the X chromosome in three Chinese indigenous sheep breeds using ovine high-density 600K single nucleotide polymorphism arrays. RESULTS: In large-tailed Han, Altay, and Tibetan sheep, respectively, a total of six, four and 22 CNV regions (CNVRs) with lengths of 1.23, 0.93, and 7.02 Mb were identified on the X chromosome. In addition, 49, 34, and 55 candidate selection regions with respective lengths of 27.49, 16.47, and 25.42 Mb were identified in large-tailed Han, Altay, and Tibetan sheep, respectively. The bioinformatics analysis results indicated several genes in these regions were associated with fat, including dehydrogenase/reductase X-linked, calcium voltage-gated channel subunit alpha1 F, and patatin like phospholipase domain containing 4. In addition, three other genes were identified from this analysis: the family with sequence similarity 58 member A gene was associated with energy metabolism, the serine/arginine-rich protein specific kinase 3 gene was associated with skeletal muscle development, and the interleukin 2 receptor subunit gamma gene was associated with the immune system. CONCLUSION: The results of this study indicated CNVRs and selection regions on the X chromosome of Chinese indigenous sheep contained several genes associated with various heritable traits.

A Transcriptomic Study of the Tail Fat Deposition in Two Types of Hulun Buir Sheep According to Tail Size and Sex.

Hulun Buir sheep of similar genetic background were divided into two lines based on tail types: Small- and big fat-tailed. To explore the molecular mechanism of fat deposition in sheep tails, we firstly evaluated the morphology and transcription level differences of tail fat between these two lines. RNA-Seq technology was used to identify differentially expressed genes (DEGs) in phenotypic extremes of tail sizes. Five comparisons were performed taking into account two factors, sex and tail type. We screened out 373 DEGs between big-tailed and small-tailed Hulun Buir sheep, and 775 and 578 DEGs between two types of tails in male and female sheep, respectively. The results showed an obvious sex difference in the fat metabolism in sheep based on gene ontology (GO), pathway, and network analyses. Intriguingly, there were two different co-expression networks only respectively shown in male and female sheep, which were insulin-related network acting on upstream pathways and PPARG-related network effect in downstream pathways. Furthermore, these two networks were linked by a classic pathway of regulating adipogenesis. This is the first study to investigate the sex differences of fat metabolism in domestic animals, and it demonstrates a new experimental way to study fat metabolism. Our findings will provide theoretical background in understanding the tail-size phenotype in sheep and can be exploited in breeding small-tailed sheep.

Advances in genome-wide association studies for important traits in sheep and goats.

Genome-wide association study (GWAS), an effective strategy to identify genetic variants associated with complex traits, has been used to study candidate genes of economical traits in animals. With the recent completion of sheep and goat genomes, single nucleotide polymorphism (SNP) chips of different densities are developed and commercialized. All these advances have enlarged the collection of molecular markers and also shed new light on the genetics of traits of interest in sheep and goats. In this review, we focus on the adoption of GWAS for important traits in sheep and goats, such as horn types, wool, dairy, growth and meat, reproduction and disease types, etc., and summarize the populations, major statistical methods and results of the GWAS analysis. Moreover, we also discuss the current state of GWAS, aiming to provide a reference for further studies on the genetic background of the important traits of sheep and goats by GWAS.

Effect of polymorphisms in the CAMKMT gene on growth traits in Ujumqin sheep.

Our previous genome-wide association study in sheep revealed that OAR3-84073899.1 (SNP31) in intron 8 of the CAMKMT gene was significantly associated with post-weaning gain at the genomic level. Herein, we performed a replication study to investigate single nucleotide polymorphisms (SNPs) within the CAMKMT gene exons, and 1000 bp of the 5'- and 3'-intranslated regions (UTRs) and their associations with growth traits in Ujumqin sheep. Five SNPs were identified through DNA pool sequencing technology: SNP26 in the 5'-UTR, SNP06 in exon 5, SNP07 in exon 8 and SNP27 and SNP28 in the 3'-UTR. Six SNPs, including SNP31 in intron 8, were genotyped in the validation group of 343 Ujumqin sheep, and each SNP was classified into three genotypes. The chi-square test suggested that all the variations were in Hardy-Weinberg equilibrium (P > 0.05) except for SNP28 and SNP31. Linkage disequilibrium analysis showed that SNP07 and SNP31 were strongly linked. An association analysis suggested that SNP06 was significantly associated with chest girth at 6 months of age (P < 0.05). SNP07 exhibited significant correlation with body weight and chest girth at 4 months of age and with body weight, chest girth and chest width at 6 months of age (P < 0.05). SNP27 was highly associated with body weight and chest girth at 4 months of age (P < 0.05), and SNP28 was extremely significantly associated with body weight and chest girth at 4 months of age and with chest girth at 6 months of age (P < 0.01). SNP31 was significantly associated with body weight and shin circumference at 4 months of age and with post-weaning gain (P < 0.05). Association analysis of the combined effect of SNP07 and SNP31 showed significant correlation with body weight and chest girth at four of months of age (P < 0.05) and with body weight and chest girth at 6 months of age (P < 0.05). These results indicate that the SNPs could be used as meritorious and available genetic markers in growth traits breeding and that the CAMKMT gene may be one of the key candidate genes that affect Ujumqin economic traits.

Identification of MEF2B and TRHDE Gene Polymorphisms Related to Growth Traits in a New Ujumqin Sheep Population.

2 SNPs were discovered in our previous genome-wide association study (GWAS): s58995.1 (rs420767326 A>G) in MEF2B gene and OAR3_115712045.1 (rs401775061 A>C) in TRHDE gene, which were significantly associated with post-weaning gain in sheep. Herein, we performed a replication experiment to investigate single nucleotide polymorphisms (SNPs) within the MEF2B and TRHDE gene exons, the 5'untranslated regions (within 1000bp), the 3' untranslated regions (within 1000bp) and their associations with Ujumqin sheep growth traits in 4-month age and 6-month age, respectively. Finally,3 SNPs were selected to be investigated including 1 SNP in 3'untranslated regions in MEF2B gene (rs417014745 A>G) and 2 SNPs in TRHDE gene (rs426980328 T>C and rs430810656 G>A).The χ2 test showed all the 3 variations were in Hardy-Weinberg equilibrium (P>0.05) status. Association analysis suggested that rs426980328 T>C was significantly associated with body weight and chest girth in 4-month age (P<0.05). rs430810656 G>A exhibited extremely significant association with body weight and chest girth in 4-month age (P<0.01). rs417014745 A>G was extremely significantly associated with body weight and chest girth in 4-month age and chest girth in 6-month age (P<0.01), and it was also significantly associated with body weight in 6-month age (P<0.05). Combined effect analysis indicated significant associations between the combinations of rs426980328-rs417014745, rs430810656-rs417014745 and several growth traits (P<0.05). These results suggested MEF2B and TRHDE genes affected growth traits in Ujumqin sheep and the combination effect of the two genes also played a significant effective role. These SNPs might have potential value as genetic markers for growth traits and it could be used in Ujumqin sheep breeding in future. Further studies are necessary to confirm our findings.

Genome-wide detection of CNVs in Chinese indigenous sheep with different types of tails using ovine high-density 600K SNP arrays.

Chinese indigenous sheep can be classified into three types based on tail morphology: fat-tailed, fat-rumped, and thin-tailed sheep, of which the typical breeds are large-tailed Han sheep, Altay sheep, and Tibetan sheep, respectively. To unravel the genetic mechanisms underlying the phenotypic differences among Chinese indigenous sheep with tails of three different types, we used ovine high-density 600K SNP arrays to detect genome-wide copy number variation (CNV). In large-tailed Han sheep, Altay sheep, and Tibetan sheep, 371, 301, and 66 CNV regions (CNVRs) with lengths of 71.35 Mb, 51.65 Mb, and 10.56 Mb, respectively, were identified on autosomal chromosomes. Ten CNVRs were randomly chosen for confirmation, of which eight were successfully validated. The detected CNVRs harboured 3130 genes, including genes associated with fat deposition, such as PPARA, RXRA, KLF11, ADD1, FASN, PPP1CA, PDGFA, and PEX6. Moreover, multilevel bioinformatics analyses of the detected candidate genes were significantly enriched for involvement in fat deposition, GTPase regulator, and peptide receptor activities. This is the first high-resolution sheep CNV map for Chinese indigenous sheep breeds with three types of tails. Our results provide valuable information that will support investigations of genomic structural variation underlying traits of interest in sheep.

Rosa26-targeted sheep gene knock-in via CRISPR-Cas9 system.

Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively easily. Technologies that facilitate specific and precise genome editing, such as knock-in, are critical for determining the functions of genes and for understanding fundamental biological processes. The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies in mammals. Rosa26 gene can encode a non-essential nuclear RNA in almost all organizations, and become a hot point of exogenous gene insertion. Here, we describe efficient, precise CRISPR/Cas9-mediated Integration using a donor vector with tGFP sequence targeted in the sheep genomic Rosa26 locus. We succeeded in integrating with high efficiency an exogenous tGFP (turboGFP) gene into targeted genes in frame. Due to its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in will become a standard method for the generation transgenic sheep.

Complete Mitochondrial Genome Sequences of Chinese Indigenous Sheep with Different Tail Types and an Analysis of Phylogenetic Evolution in Domestic Sheep.

China has a long history of sheep (Ovis aries [O. aries]) breeding and an abundance of sheep genetic resources. Knowledge of the complete O. aries mitogenome should facilitate the study of the evolutionary history of the species. Therefore, the complete mitogenome of O. aries was sequenced and annotated. In order to characterize the mitogenomes of 3 Chinese sheep breeds (Altay sheep [AL], Shandong large-tailed sheep [SD], and small-tailed Hulun Buir sheep [sHL]), 19 sets of primers were employed to amplify contiguous, overlapping segments of the complete mitochondrial DNA (mtDNA) sequence of each breed. The sizes of the complete mitochondrial genomes of the sHL, AL, and SD breeds were 16,617 bp, 16,613 bp, and 16,613 bp, respectively. The mitochondrial genomes were deposited in the GenBank database with accession numbers KP702285 (AL sheep), KP981378 (SD sheep), and KP981380 (sHL sheep) respectively. The organization of the 3 analyzed sheep mitochondrial genomes was similar, with each consisting of 22 tRNA genes, 2 rRNA genes (12S rRNA and 16S rRNA), 13 protein-coding genes, and 1 control region (D-loop). The NADH dehydrogenase subunit 6 (ND6) and 8 tRNA genes were encoded on the light strand, whereas the rest of the mitochondrial genes were encoded on the heavy strand. The nucleotide skewness of the coding strands of the 3 analyzed mitogenomes was biased toward A and T. We constructed a phylogenetic tree using the complete mitogenomes of each type of sheep to allow us to understand the genetic relationships between Chinese breeds of O. aries and those developed and utilized in other countries. Our findings provide important information regarding the O. aries mitogenome and the evolutionary history of O. aries inside and outside China. In addition, our results provide a foundation for further exploration of the taxonomic status of O. aries.

Detection of Selection Signatures on the X Chromosome in Three Sheep Breeds.

Artificial selection has played a critical role in animal breeding. Detection of artificial selection footprints in genomic regions can provide insights for understanding the function of specific phenotypic traits and better guide animal breeding. To more fully understand the relationship between genomic composition and phenotypic diversity arising from breed development, a genome-wide scan was conducted using an OvineSNP50 BeadChip and integrated haplotype score and fixation index analyses to detect selection signatures on the X chromosome in three sheep breeds. We identified 49, 34, and 55 candidate selection regions with lengths of 27.49, 16.47, and 25.42 Mb in German Mutton, Dorper, and Sunit sheep, respectively. Bioinformatics analysis showed that some of the genes in these regions with selection signatures, such as BMP15, were relevant to reproduction. We also identified some selection regions harboring genes that had human orthologs, including BKT, CENPI, GUCY2F, MSN, PCDH11X, PLP1, VSIG4, PAK3, WAS, PCDH19, PDHA1, and SRPX2. The VSIG4 and PCDH11X genes are associated with the immune system and disease, PDHA1 is associated with biosynthetic related pathways, and PCDH19 is expressed in the nervous system and skin. These genes may be useful as candidate genes for molecular breeding.

Cloning and expression of MyoG gene from Hu sheep and identification of its myogenic specificity.

This experiment was conducted to explore the biological functions of myogenin (MyoG) gene. MyoG gene was cloned from genome of Hu sheep by overlap extension PCR. Then, pEGFP-C1-MyoG and pcDNA3.0-MyoG fusion expression vectors was constructed and pEGFP-C1-MyoG vector had been transfected into NIH-3T3 cells by liposomes-mediated method, and MyoG was detected in vitro by RT-PCR,western blotting and its subcellular localization by EGFP marker. pcDNA3.0-MyoG was transfected into goat embryonic fibroblasts (GEF) cells in order to detect the myogenic function of MyoG in vitro. Then pEGFP-C1-MyoG plasmid was injected into the testes of sheep and goat, respectively, to produce the transgenic generation. The results showed that the length of MyoG coding region of Hu sheep was 675 bp, encoding 224 amino acids. Compared with goat, cattle, pig and rat, the sequence homology of sheep MyoG cDNA was 99.26, 97.04, 92.00, and 87.70 %, respectively. The bioinformatics prediction showed that MyoG protein contained a typical bHLH structure, but without a short signal peptide, revealing that MyoG protein might be a non-secretory protein. The result of RT-PCR and western blotting demonstrated that MyoG could be expressed successfully in the transfected cells in vitro and the MyoG protein was located in nucleus. The positive transfected GEF cells with pcDNA3.0-MyoG were found to express desmin protein. The positive rates of transgenic sheep and transgenic goat were 7.1 and 7.4 % in F1 generation, respectively. Conclusively, MyoG cDNA from Hu sheep had been cloned successfully. The subcellular localization and myogenic activity of MyoG were exactly detected on the basis of multiple biological analyses, which expanded our understanding of the biological function of MyoG.

Cloning of the Xuhuai goat PPARγ gene and the preparation of transgenic sheep.

This study aimed to clone the peroxisome proliferator-activated receptor γ (PPARγ) gene of the Xuhuai goat and to make transgenic sheep using intratesticular injection, so as to improve the meat quality and flavor by increasing the intramuscular fat content. The coding sequence of the goat PPARγ gene was 1,428 bp, encoding 475 amino acids. Its similarity with other species was 81 (chicken), 89 (mouse), 92 (pig), 98 (cow), and 99% (sheep). The similarity of the corresponding amino acid sequences was 92.9, 97.3, 98.3, 99.6, and 99.8%, respectively. The signal peptide region of the PPARγ protein was not found in this study, demonstrating that the protein is not secreted. RT-PCR and western blot revealed that PPARγ was expressed in vitro, and the protein was localized in the cytoplasm. The PPARγ gene was expressed in F1 transgenic sheep at both the mRNA and the protein levels; the positive ratio was 13.7%.

Production of transgenic mice expressing the goat H-FABP gene by intratesticular injection.

The objective of this study was to explore the possibility of obtaining stable transgenic animals by intratesticular injection. The recombinant vector pEGFP-H-FABP expressing the goat heart-type fatty acid binding protein and green fluorescent protein was mixed with liposome complexes and randomly injected into the testes of mice. Testicular section, fluorescence, and DNA detection assays of mouse sperm were performed to determine the integration of foreign DNA. The results showed that foreign DNA was successfully expressed in the treated mice. Furthermore, the expression and function of the foreign gene were analyzed in F1 generation and F2 generation mice at different levels, with the positive rates of foreign gene transfer into the F1 and F2 generations being 4.0 and 30.23 %, respectively. These results strongly support testicular injection as an effective method of producing transgenic animals and indicate that foreign genes can be stably passed on to the offspring. This research has theoretical and practical implications for the improvement in the quality of laboratory animals and for gene therapy.

Cloning of Xuhuai goat lipoprotein lipase gene and the preparation of transgenic sheep.

This paper presents cloning of cDNA of lipoprotein lipase (LPL) gene from Xuhuai goat, and the sub-cellular localization analysis through enhanced green fluorescent (EGFP) fusion protein. cDNA was cloned by reverse transcription polymerase chain reaction (RT-PCR). Fusion expression vector named pEGFP-LPL was constructed successfully. Then NIH-3T3 cells were transfected with pEGFP-LPL through polyethylene imine and observed under inverted microscope after 48 h transfection. The RT-PCR was performed to analysis the level of expression of mRNA. The complete coding sequence (1,530 bp) of LPL was acquired, and the open reading frame size was 1,437 bp with a capacity to encode 478 amino acids. The prediction of signal peptide region showed that LPL protein contained a short signal peptide with a probability of 100 %, and the signal peptidase cleavage site located between the 23rd and the 24th amino acid with a probability of 65.9 %. RT-PCR results showed the LPL mRNA expressed successfully in vitro. Sub-cellullar localization analysis showed that pEGFP-LPL fusion protein located at the cytoplasm. LPL gene of Xuhuai goat was transfer into sheep by testicular injection. According to detection from different level, the LPL gene was expressed successfully in F(1) generation.

[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.

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.