Molecular survey and genetic identification of Anaplasma species in goats from central and southern China

Anaplasma species are obligate intracellular rickettsial pathogens that impact the health of humans and animals. Few studies have been carried out on Anaplasma infections in central and southern China. This study was conducted to determine the coinfection rates of Anaplasma ovis, A. bovis, and A. phagocytophilum from 262 field blood samples of goats in these regions. The average prevalences of single infection of A. ovis, A. bovis, and A. phagocytophilum were 15.3, 16.0, and 6.1%, respectively. Coinfection of A. ovis and A. bovis was dominant, with an infection rate of 27.1%. Coinfection of A. ovis and A. phagocytophilum was 1.9% and that of A. bovis and A. phagocytophilum was 4.2%. Three-pathogen coinfection was found in three of four investigated provinces with a prevalence between 0 and 5.3%. The accuracy of the PCR results was corroborated by sequencing. Analysis of the 16S rRNA gene sequences of A. bovis and A. phagocytophilum confirmed the presence of these pathogens at the investigated sites and indicated the possible genetic diversity of A. phagocytophilum. Field blood inoculation of experimental animals led to successful identification and observation of the morphological shapes of A. bovis in the infected monocytes of sheep. Phylogenetic study with msp4 sequences of A. ovis indicated that the A. ovis genotypes from sheep in the north differed from the genotypes of goats in the investigated sites.     

Bacterial diversity in worker adults of Apis mellifera capensis and Apis mellifera scutellata (Insecta: Hymenoptera) assessed using 16S rRNA sequences

High-fidelity PCR of 16S rRNA sequences was used to identify bacteria associated with worker adults of the honeybee subspecies Apis mellifera capensis and Apis mellifera scutellata. An expected approximately 1.5-kb DNA band, representing almost the entire length of the 16S rRNA gene, was amplified from both subspecies and cloned. Ten unique sequences were obtained: one sequence each clustered with Bifidobacterium (Gram-positive eubacteria), Lactobacillus (Gram-positive eubacteria), and Gluconacetobacter (Gram-negative alpha-proteobacteria); two sequences each clustered with Simonsiella (beta-proteobacteria) and Serratia (gamma-proteobacteria); and three sequences each clustered with Bartonella (alpha-proteobacteria). Although the sequences relating to these six bacterial genera initially were obtained from either A. m. capensis or A. m. scutellata or both, newly designed honeybee-specific 16S rRNA primers subsequently amplified all sequences from all individual workers of both subspecies. Attempts to amplify these sequences from eggs have failed. However, the wsp primers designed to amplify Wolbachia DNA from arthropods, including these bees, consistently produced a 0.6-kb DNA band from individual eggs, indicating that amplifiable bacterial DNA was present. Hence, the 10 bacteria could have been acquired orally from workers or from other substrates. This screening of 16S rRNA sequences from A. m. capensis and A. m. scutellata found sequences related to Lactobacillus and Bifidobacterium which previously had been identified from other honeybee subspecies, as well as sequences related to Bartonella, Gluconacetobacter, Simonsiella/Neisseria, and Serratia, which have not been identified previously from honeybees.     

Phylogenetic Analysis of Ruminant Theileria spp. from China Based on 28S Ribosomal RNA Gene

Species identification using DNA sequences is the basis for DNA taxonomy. In this study, we sequenced the ribosomal large-subunit RNA gene sequences (3,037-3,061 bp) in length of 13 Chinese Theileria stocks that were infective to cattle and sheep. The complete 28S rRNA gene is relatively difficult to amplify and its conserved region is not important for phylogenetic study. Therefore, we selected the D2-D3 region from the complete 28S rRNA sequences for phylogenetic analysis. Our analyses of 28S rRNA gene sequences showed that the 28S rRNA was useful as a phylogenetic marker for analyzing the relationships among Theileria spp. in ruminants. In addition, the D2-D3 region was a short segment that could be used instead of the whole 28S rRNA sequence during the phylogenetic analysis of Theileria, and it may be an ideal DNA barcode.     

Orientobilharzia turkestanicum is a member of Schistosoma genus based on phylogenetic analysis using ribosomal DNA sequences

In the present study, samples representing Orientobilharzia turkestanicum from cattle, sheep, cashmere goat and goat in Heilongjiang Province, China, were characterized and grouped genetically by sequences of internal transcribed spacer (ITS, including ITS-1 and ITS2) and 28S ribosomal DNA (28S rDNA). The ITS and 28S rDNA were amplified by polymerase chain reaction (PCR) and then sequenced and compared with that of other members of the Schistosomatidae available in GenBank™, and phylogenetic relationships between them were re-constructed using the neighbor-joining and maximum parsimony methods. The lengths of ITS-1, ITS-2 and 28S rDNA sequences for all O. turkestanicum samples from different hosts were 384 bp, 331 bp and 1304 bp, respectively. While the ITS-1 sequences of O. turkestanicum from each of the four different hosts, and ITS-2 of O. turkestanicum from cattle, sheep and cashmere goat were identical, respectively, the ITS-2 of O. turkestanicum from goat differed from that of O. turkestanicum from cattle, sheep and cashmere goat by one nucleotide. The 28S rDNA sequences of O. turkestanicum from sheep and cashmere goat were identical, but differed from that of O. turkestanicum from cattle and goat by two nucleotides, with the latter two also having identical 28S rDNA sequence. Phylogenetic analyses based on the combined sequences of the ITS-1 and ITS-2, or the 28S rDNA sequences placed O. turkestanicum within the genus Schistosoma, and it was phylogenetically closer to the African schistosome group than to the Asian schistosome group. These results should have implications for studying the origin and evolution of O. turkestanicum and other members of the Schistosomatidae.     

Study of the heterogeneity of 16s rRNA gene and groESL operone in the dna samples of Anaplasma phagocytophilum, Ehrlichia muris, and "Candidatus Neoehrlichia mikurensis" determined in the Ixodes persulcatus ticks in the area of Urals, Siberia, and far east of Russia

A total of 3552 Ixodes persulcatus from Sverdlovsk, Chelyabinsk, Novosibirsk, Irkutsk regions and Khabarovsk Territory were examined on the Ehrlichia and Anaplasma presence by nested PCR based on the 16S rRNA gene. Both Anaplasma phagocytophilum and Ehrlichia muris DNA were found in I. persulcatus in all studied regions. A. Phagocytophilum was detected in 1.3-6.3% of ticks and E. muris - in 2.0-14.1% of ticks. Moreover, "Candidatus Neoehrlichia mikurensis" DNA was found in 8 ticks collected in Novosibirsk, Irkutsk Regions and Khabarovsk Territory. Partial nucleotide sequences of 16S rRNA gene and groESL operone (1240-1300 bp) were determined for 65 samples of A. Phagocytophilum, 17 samples of E. muris and 4 samples of "Candidatus Neoehrlichia mikurensis". Nucleotide sequences of 16S rRNA gene and groESL operone of E. muris and "Candidatus Neoehrlichia mikurensis" were shown to be highly conservative, and nucleotide sequences of groESL operone of both E. muris and "Candidatus Neoehrlichia mikurensis" differed from the sequences found previously in other species of Ixodid tick. On the basis of analysis of the 16S rRNA gene and groESL operone sequences it was concluded that all revealed samples A. Phagocytophilum could be divided into 2 groups. GroESL operone sequences of A. Phagocytophilum from the first group were identical to each other but significantly differed from the known groESL operone sequences (less than 98.2% of similarity), whereas their 16S rRNA gene sequences were identical to the sequence of widely distributed and pathogenic for human A. Phagocytophilum genetic variant (CAHU-HGEl, GenBank AF093788) or differed from it by a single nucleotide substitution. The nucleotide sequences of groESL operone of A. Phagocytophilum from the second group differed from each other by 1-4 nucleotides and were closely related (99.2-99.4% of similarity) to the sequences of groESL operone ofA. phagocytophilum isolates found in Europe in Ixodes ricinus and roe deer. The nucleotide sequences of the 16S rRNA gene of A. Phagocytophilum from the second group were most similar to the sequence of the rare A. Phagocytophilum genetic variant previously found only in China (GenBank DQ342324).     

Genetic Diversity of Echinococcus granulosus in Center of Iran

Hydatid cyst caused by Echinococcus granulosus is one of the most important parasitic diseases around the world and many countries in Asia, including Iran, are involved with this infection. This disease can cause high mortality in humans as well as economic losses in livestock. To date, several molecular methods have been used to determine the genetic diversity of E. granulosus. So far, identification of E. granulosus using real-time PCR fluorescence-based quantitative assays has not been studied worldwide, also in Iran. Therefore, the aim of this study was to investigate the genetic diversity of E. granulosus from center of Iran using real-time PCR method. A total of 71 hydatid cysts were collected from infected sheep, goat, and cattle slaughtered in Isfahan, Iran during 2013. DNA was extracted from protoscolices and/or germinal layers from each individual cyst and used as template to amplify the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) (420 bp). Five cattle isolates out of 71 isolates were sterile and excluded from further investigation. Overall, of 66 isolates, partial sequences of the cox1 gene of E. granulosus indicated the presence of genotypes G1 in 49 isolates (74.2%), G3 in 15 isolates (22.7%), and G6 in 2 isolates (3.0%) in infected intermediate hosts. Sixteen sequences of G1 genotype had microgenetic variants, and they were compared to the original sequence of cox1. However, isolates identified as G3 and G6 genotypes were completely consistent with original sequences. G1 genotype in livestock was the dominant genotype in Isfahan region, Iran.     

Anaplasma phagocytophilum in small mammals and ticks in northeast Florida

Human anaplasmosis is an emerging tick-borne disease in the United States, but few studies of the causative agent, Anaplasma phagocytophilum, have been conducted in southeastern states. The aim of this study was to determine if A. phagocytophilum is present in small mammals and ticks in northeast Florida. Polymerase chain reaction assays designed to amplify portions of the major surface protein 2 gene (p44), 16S rDNA, and groESL operons were used to test rodent blood and tick DNA samples for the presence of A. phagocytophilum. Positive samples were confirmed by DNA sequence analysis. Anaplasma phagocytophilum DNA was detected in less than 5% of cotton mice and 45% of cotton rats from two sites in northeast Florida. Anaplasma phagocytophilum DNA was also confirmed in 1.3% of host-seeking adult Ixodes scapularis tested and 2.7% of host-seeking adult Amblyomma americanum. This report describes the first DNA sequence data confirming strains of A. phagocytophilum in rodents and ticks in Florida. The DNA sequences of the msp2, 16S rDNA, and groESL gene fragments obtained in this study were highly similar to reference strains of human pathogenic strains of A. phagocytophilum. These findings suggest that A. phagocytophilum is present and established among some small mammal species in northeast Florida. Although the infection prevalence was low in the total number of ticks tested, the presence of A. phagocytophilum in two human biting tick species, one of which is a known competent vector, suggests that humans in this region may be at risk of granulocytic anaplasmosis caused by this pathogen.     

Phylogenetic analysis of rumen bacteria by comparative sequence analysis of cloned 16S rRNA genes

Comparative DNA sequence analysis of 16S rRNA genes (rDNA) was undertaken to further our understanding of the make-up of bacterial communities in the rumen fluid of dairy cattle. Total DNA was extracted from the rumen fluid of 10 cattle fed haylage/corn silage/concentrate rations at two different times. Rumen samples were collected on two separate occasions from five cows each. In experiment 1, 31 cloned rDNA sequences were analysed. In experiment 2, DNA extractions were amplified using either 12 or 30 cycles of PCR in order to examine biases introduced during the reactions. A set of 53 sequences were analysed in experiment 2 from DNA amplified using 12 cycles and 49 sequences from PCR using 30 cycles. Sequences from the 5' end of 16S rRNA gene were compared with existing sequences in the Ribosomal Database Project. Clones from experiment 1 produced a data set in which 55% of the sequences were similar to low G+C Gram-positive bacteria related to the genus Clostridia, the majority of which were closely related to bacteria in Cluster XIV. Approximately 30% of the cloned sequences were related to bacteria in the Prevotella-Bacteroides group. Clones from experiment 2 produced a data set in which the majority of sequences were related to the Prevotella-Bacteroides group, regardless of the number of cycles of PCR. The remaining sequences clustered with members of the genus Clostridia. The majority of rDNA sequences analysed in this study represent novel rumen bacteria which have not yet been isolated.     

16S rRNA和recA、groEL基因分类鉴定乳酸乳球菌乳酸亚种和乳脂亚种的比较

【目的】比较16S rRNA和recA、groEL基因部分序列用于乳酸乳球菌乳酸亚种和乳脂亚种分类鉴定的效果。【方法】对已鉴定的8株分离自传统发酵乳的乳酸乳球菌, 选取recA和groEL基因片段, 通过PCR扩增、测序, 将测序得到的序列比对后构建系统发育树, 并与16S rRNA基因序列分析技术进行比较。【结果】比较分析不同菌株16S rRNA和recA、groEL基因的亲缘关系, recA、groEL基因可以准确地完成乳酸乳球菌乳酸亚种和乳脂亚种的区分和鉴定。【结论】recA和groEL基因序列分析可以实现乳酸乳球菌乳酸亚种和乳脂亚种的区分, 因其具有快速、准确、稳定的特点, 可适合于乳酸乳球菌乳酸亚种和乳脂亚种间的快速分类鉴定。     

Genetic characterization of Anaplasma ovis strains from bighorn sheep in Montana

Wildlife reservoir species and genetic diversity of Anaplasma ovis (Rickettsiales: Anaplasmataceae) have been poorly characterized. Bighorn sheep (Ovis canadensis), captured in Montana from December 2004 to January 2005, were tested for antibodies to Anaplasma spp.; the presence of A. ovis was determined by the characterization of major surface protein msp4 sequences. Anaplasma antibodies were detected in 25/180 (14%) sampled bighorn sheep and A. ovis msp4 sequences were amplified by polymerase chain reaction (PCR) and sequenced from 9/23 (39%) of seropositive animals. All animals were negative by PCR for the related pathogens, Anaplasma phagocytophilum and Anaplasma marginale. All msp4 sequences identified in the bighorn sheep were identical and corresponded to a single A. ovis genotype that was identical to a sheep isolate reported previously from Idaho. The finding of a single genotype of A. ovis in this wild herd of bighorn sheep was in contrast to the genetic diversity reported for A. marginale in cattle herds in the western United States and worldwide. These results demonstrated that bighorn sheep may be a wildlife reservoir of A. ovis in Montana.     

Comparisons of Isolates of Heterodera avenae using 2-D PAGE Protein Patterns and Ribosomal DNA

Six geographic isolates of Heterodera avenae, including two isolates each from Sweden, Australia, and the United States, were compared on the basis of 2-D PAGE protein patterns and the complete DNA sequence for the two internal transcribed ribosomal DNA spacers (rDNA ITS1 and ITS2) and the 5.8S rRNA gene. The protein pattern data and rDNA ITS sequence data both indicated that the Swedish Gotland strain of H. avenae differed markedly from the rest of the isolates. Protein patterns for the Australia isolates differed more from a Swedish strict H. avenae isolate and isolates from Oregon and Idaho, than the two U.S. isolates and the Swedish strict H. avenae isolate differed from each other. Except for the Gotland strain isolate, the rDNA ITS sequences were highly conserved among all of the H. avenae isolates, just as we earlier found them to be conserved among species of the schachtii group of Heterodera.     

成年核移植山羊生长相关基因的表达分析

体细胞核移植过程有可能影响克隆动物生长相关基因尤其是印迹基因的表达水平。本研究运用同源引物 PCR 扩增、RACE 技术并结合同源克隆策略, 克隆了 7 个山羊生长相关基因包括 3 个印迹基因(H19、IGF2 和 IGF2R)和 4 个非印迹基因(IGF1、IGF1R、GHR 和 GHSR)的完全 CDS 或者部分 cDNA 序列, 经生物信息学技术确认后, 用荧光实时定量 PCR 对 8只成年克隆山羊中这些基因的表达水平进行分析, 结果表明 3 个印迹基因中 IGF2R 基因表达水平极显著高于对照组的自然繁殖山羊(P<0.01), 而 H19 和 IGF2 的表达则没有很大区别; 4 个非印迹基因中只有 IGF1R 的表达水平极显著高于对照组(P<0.01), IGF1、GHR 和 GHSR 的表达与对照组相似。表明即使在表型正常的成年克隆动物也存在一定的表观遗传异常。通过对获得完全 CDS 和 3′UTR 的 IGF2 基因经过生物信息学分析表明, 山羊 IGF2 基因包含一个 540 bp 的开放阅读框 (ORF)编码 179 个氨基酸。IGF2 基因 cDNA 序列和氨基酸序列以及其它基因部分序列比较分析表明, 山羊所有这些基因与绵羊的同源性要高于同牛的同源性。     

Molecular Characterization of Taenia multiceps Isolates from Gansu Province,China by Sequencing of Mitochondrial Cytochrome C Oxidase Subunit 1

A total of 16 Taenia multiceps isolates collected from naturally infected sheep or goats in Gansu Province, China were characterized by sequences of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. The complete cox1 gene was amplified for individual T. multiceps isolates by PCR, ligated to pMD18T vector, and sequenced. Sequence analysis indicated that out of 16 T. multiceps isolates 10 unique cox1 gene sequences of 1,623 bp were obtained with sequence variation of 0.12-0.68%. The results showed that the cox1 gene sequences were highly conserved among the examined T. multiceps isolates. However, they were quite different from those of the other Taenia species. Phylogenetic analysis based on complete cox1 gene sequences revealed that T. multiceps isolates were composed of 3 genotypes and distinguished from the other Taenia species.     

Sequence of specific mitochondrial 16S rRNA gene fragment from Egyptian buffalo is used as a pattern for discrimination between river buffaloes,cattle, sheep and goats

Characterization of molecular markers and the development of better assays for precise and rapid detection of domestic species are always in demand. This is particularly due to recent food scares and the crisis of biodiversity resulting from the huge ongoing illegal traffic of endangered species. The aim of this study was to develop a new and easy method for domestic species identification (river buffalo, cattle, sheep and goat) based on the analysis of a specific mitochondrial nucleotide sequence. For this reason, a specific fragment of Egyptian buffalo mitochondrial 16S rRNA gene (422 bp) was amplified by PCR using two universal primers. The sequence of this specific fragment is completely conserved between all tested Egyptian buffaloes and other river buffaloes in different places in the world. Also, the lengths of the homologous fragments were less by one nucleotide (421 bp) in case of goats and two nucleotides (420 bp) in case of both cattle and sheep. The detection of specific variable sites between investigated species within this fragment was sufficient to identify the biological origin of the samples. This was achieved by alignment between the unknown homologous sequence and the reference sequences deposited in GenBank database (accession numbers, FJ748599–FJ748607). Considering multiple alignment results between 16S rRNA homologous sequences obtained from GenBank database with the reference sequence, it was shown that definite nucleotides are specific for each of the four studied species of the family Bovidae. In addition, other nucleotides are detected which can allow discrimination between two groups of animals belonging to two subfamilies of family Bovidae, Group one (closely related species like cattle and buffalo, Subfamily Bovinae) and Group two (closely related species like sheep and goat, Subfamily Caprinae). This 16S DNA barcode character-based approach could be used to complement cytochrome c oxidase I (COI) in DNA barcoding. Also, it is a good tool for identification of unknown sample belonging to one of the four domestic animal species of family Bovidae quickly and easily.     

The phylogenetic significance of peptidoglycan types: Molecular analysis of the genera Microbacterium and Aureobacterium based upon sequence comparison of gyrB, rpoB, recA and ppk and 16SrRNA genes

The type strains of 27 species of the genus Microbacterium, family Microbacteriaceae, were analyzed with respect to the phylogeny of the housekeeping genes coding for DNA gyrase subunit B (gyrB), RNA-polymerase subunit B (rpoB), recombinase A (recA) and polyphosphate kinase (ppk). The resulting gene trees were compared to the 16S rRNA gene phylogeny of the same species. The topology of neighbour-joining and maximum parsimony phylogenetic trees based upon nucleic acid sequences and protein sequences of housekeeping genes differed among each other and no gene tree was identical to that of the 16S rRNA gene tree. Only some species showed consistent clustering by all genes analyzed, but the majority of species branched with different neighbours in most gene trees. The failure to phylogenetically cluster type strains into two groups based upon differences in the amino acid composition of peptidoglycan on the basis of 16S rRNA gene sequence similarity, once leading to the union of the genera Microbacterium and Aureobacterium, was also seen in the analysis of recA, rpoB and gyrB gene and protein phylogenies. Analysis of the pkk gene and protein as well as of a concatenate tree, combining sequences of all five genes (total of 3.700 nucleotides), sees members of the former genus Aureobacterium and other type strains with lysine as diagnostic diamino acid to form a coherent cluster that branches within the radiation of Microbacterium species with ornithine in the peptidoglycan.     

Polymorphism in exon 3 of follicle stimulating hormone beta (FSHB) subunit gene and its association with litter traits and superovulation in the goat

The polymorphism in follicle stimulating hormone beta (FSHB) subunit gene was detected by PCR-SSCP and PCR-RFLP methods in 780 does from four goat breeds including Boer, Matou, Black and Boer-Matou crossbred (BM). The associations of FSHB genotypes with litter size (LS), litter weight at birth (LWB) and gestation length (GL) as well as superovulation performances were analyzed in relatively lower-prolific Boer and higher-prolific Matou breeds. A 1514 bp linear DNA sequence of goat FSHB gene covering the complete 3 exons was cloned by four pairs of primer. A new mutation (A2645G, GenBank Accession no: S64745) locating in exon 3 causing an amino acid change from glutamine (Gln) to arginine (Arg) at residue 115 was identified, which resulted in three genotypes named AA, AB and BB. The three genotypes were detected in the four studied goat breeds. The higher-prolific Matou breed had the highest frequency of genotype AA. Association analysis showed that Boer and Matou does with AA genotype have the largest LS both in average and in parities from first to fourth (P < 0.05). Furthermore, Boer does with AA genotype had the heaviest LWB compared to other two genotypes (P < 0.05). Matou does with AA genotype have more numbers of ova harvested, large follicles and corpus lutea on ovaries after superovulation than those with BB genotype (P < 0.05). Therefore, these results suggest that the FSHB gene is a candidate gene that affects reproduction traits in goats.     

Sequence Analysis of the <Emphasis Type="Italic">Lactoferrin</Emphasis> Gene and Variation of <Emphasis Type="Italic">g</Emphasis>.<Emphasis Type="Italic">7605C</Emphasis>→<Emphasis Type="Italic">T</Emphasis> in 10 Chinese Indigenous Goat Breeds

Much attention has been focused on the study of lactoferrin at the protein or nucleotide level in mice, humans, and cattle, but little is known about it in goats. The goat LF gene from 5' UTR to exon 17 was amplified, and the variation of g.7605C→T in 10 Chinese indigenous goat breeds was analyzed. Among the three ruminant species (cattle, sheep, and goats), the intron-exon distribution pattern was similar, and all the exons had the same length, but the length of introns varied greatly due to insertions or deletions. The frequency of allele T at g.7605C→T (50.12%) was a little higher than that of allele C (49.88%), and the genotype distribution differed greatly between goat populations. The g.7605C→T site showed higher genetic diversity in goat populations. The genetic differentiation was 0.0783, and gene flow was 2.9433 among the 10 Chinese indigenous goat populations.     

Identification of msp1 Gene Variants in Populations of Meloidogyne incognita Using PCR-DGGE

Effectors of root-knot nematodes are essential for parasitism and prone to recognition by adapted variants of the host plants. This selective pressure initiates hypervariability of effector genes. Diversity of the gene variants within nematode populations might correlate with host preferences. In this study we developed a method to compare the distribution of variants of the effector gene msp1 among populations of Meloidogyne incognita. Primers were designed to amplify a 234-bp fragment of msp1. Sequencing of cloned PCR products revealed five msp1 variants from seven populations that were distinguishable in their reproduction on five host plants. A protocol for denaturing gradient gel electrophoresis (DGGE) was developed to separate these msp1 variants. DGGE for replicated pools of juveniles from the seven populations revealed ten variants of msp1. A correlation between the presence of a particular gene variant and the reproductive potential on particular hosts was not evident. Especially race 3 showed substantial variation within the population. DGGE fingerprints of msp1 tended to cluster the populations according to their reproduction rate on pepper. The developed method could be useful for analyzing population heterogeneity and epidemiology of M. incognita.     

Targeting the exogenous htPAm gene on goat somatic cell beta-casein locus for transgenic goat production

Combining gene targeting of animal somatic cells with nuclear transfer technique has provided a powerful method to produce transgenic animal mammary gland bioreactor. The objective of this study is to make an efficient and reproducible gene targeting in goat fetal fibroblasts by inserting the exogenous htPAm cDNA into the beta-casein locus with liposomes or electroporation so that htPAm protein might be produced in gene-targeted goat mammary gland. By gene-targeting technique, the exogenous htPAm gene was inserted to milk goat beta-casein gene sequences. Fetal fibroblasts were isolated from Day 35 fetuses of Guanzhong milk goats, and transfected with linear gene-targeting vector pGBC4htPAm using Lipefectamin-2000 and electoporation, respectively. Forty-eight gene-targeted cell colonies with homologous recombination were obtained, and three cell colonies were verified by DNA sequence analysis within the homologous recombination region. Using gene-targeted cell lines as donor cells for nuclear transfer, a total of 600 reconstructed embryos had been obtained, and 146 developed cloned embryos were transferred to 16 recipient goats, and finally three goats showed pregnancy at Day 90.