Association of functional sequence variants of the myosin heavy chain 3 gene with muscle collagen content in pigs.

This study examined the association between functional sequence variants (FSVs) of myosin heavy chain 3 (MYH3) genotypes and collagen content in a Landrace and Jeju native pig (JNP) crossbred population. Four muscles (Musculus longissimus dorsi, Musculus semimembranosus, Musculus triceps brachii, and Musculus biceps femoris) were used for the analysis of meat collagen content, and the same animals were genotyped for the FSVs of the MYH3 gene by using PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism). Three FSVs of MYH3 genotypes were identified and had genotype frequencies of 0.358, 0.551, and 0.091 for QQ, Qq, and qq, respectively. QQ animals for the FSVs of the MYH3 genotypes showed higher collagen content in their M. longissimus dorsi (p < 0.001), M. semimembranosus (p < 0.001), M. triceps brachii (p < 0.001), and M. biceps femoris (p < 0.001) than qq homozygous animals. After the validation of this result in other independent populations, the FSVs of MYH3 genotypes can be a valuable genetic marker for improving collagen content in porcine muscles and can also be applied to increase the amount of collagen for biomedical purposes.

Identification of Copy Number Variations in Four Horse Breed Populations in South Korea.

In this study, genome-wide CNVs were identified using a total of 469 horses from four horse populations (Jeju horses, Thoroughbreds, Jeju riding horses, and Hanla horses). We detected a total of 843 CNVRs throughout all autosomes: 281, 30, 301, and 310 CNVRs for Jeju horses, Thoroughbreds, Jeju riding horses, and Hanla horses, respectively. Of the total CNVRs, copy number losses were found to be the most abundant (48.99%), while gains and mixed CNVRs accounted for 41.04% and 9.96% of the total CNVRs, respectively. The length of the CNVRs ranged from 0.39 kb to 2.8 Mb, while approximately 7.2% of the reference horse genome assembly was covered by the total CNVRs. By comparing the CNVRs among the populations, we found a significant portion of the CNVRs (30.13%) overlapped; the highest number of shared CNVRs was between Hanla horses and Jeju riding horses. When compared with the horse CNVRs of previous studies, 26.8% of CNVRs were found to be uniquely detected in this study. The CNVRs were not randomly distributed throughout the genome; in particular, the Equus caballus autosome (ECA) 7 comprised the largest proportion of its genome (16.3%), while ECA 24 comprised the smallest (0.7%). Furthermore, functional analysis was applied to CNVRs that overlapped with genes (genic-CNVRs); these overlapping areas may be potentially associated with the olfactory pathway and nervous system. A racing performance QTL was detected in a CNVR of Thoroughbreds, Jeju riding horses, and Hanla horses, and the CNVR value was mixed for three breeds.

GWAS and Post-GWAS High-Resolution Mapping Analyses Identify Strong Novel Candidate Genes Influencing the Fatty Acid Composition of the Longissimus dorsi Muscle in Pigs.

Fatty acid (FA) composition is one of the most important parameters for the assessment of meat quality in pigs. The FA composition in pork can also affect human health. Our aim was to identify quantitative trait loci (QTLs) and positional candidate genes affecting the FA profile of the longissimus dorsi muscle in a large F2 intercross between Landrace and Korean native pigs comprising 1105 F2 progeny by genome-wide association studies (GWAS) and post-GWAS high-resolution mapping analyses. We performed GWAS using the PorcineSNP60K BeadChip and a linear mixed model. Four genome-wide significant QTL regions in SSC8, SSC12, SSC14, and SSC16 were detected (p < 2.53 × 10-7). Several co-localizations of QTLs in SSC12 for oleic acid, linoleic acid, arachidonic acid, monounsaturated FAs, polyunsaturated FAs, and the polyunsaturated/saturated FA ratio were observed. To refine the QTL region in SSC12, a linkage and linkage disequilibrium analysis was applied and could narrow down the critical region to a 0.749 Mb region. Of the genes in this region, GAS7, MYH2, and MYH3 were identified as strong novel candidate genes based on further conditional association analyses. These findings provide a novel insight into the genetic basis of FA composition in pork and could contribute to the improvement of pork quality.

Genome-wide analyses of the Jeju, Thoroughbred, and Jeju crossbred horse populations using the high density SNP array.

The Jeju horse is an indigenous Korean horse breed that is currently registered with the Food and Agriculture Organization of the United Nations. However, there is severe lack of genomic studies on Jeju horse. This study was conducted to investigate genetic characteristics of horses including Jeju horse, Thoroughbred and Jeju crossbred (Jeju × Thoroughbred) populations. We compared the genomes of three horse populations using the Equine SNP70 Beadchip array. Short-range Linkage disequilibrium was the highest in Thoroughbred, whereas r2 values were lowest in Jeju horse. Expected heterozygosity was the highest in Jeju crossbred (0.351), followed by the Thoroughbred (0.337) and Jeju horse (0.311). The level of inbreeding was slightly higher in Thoroughbred (- 0.009) than in Jeju crossbred (- 0.035) and Jeju horse (- 0.038). FST value was the highest between Jeju horse and Thoroughbred (0.113), whereas Jeju crossbred and Thoroughbred showed the lowest value (0.031). The genetic relationship was further assessed by principal component analysis, suggesting that Jeju crossbred is more genetically similar to Thoroughbred than Jeju horse population. Additionally, we detected potential selection signatures, for example, in loci located on LCORL/NCAPG and PROP1 genes that are known to influence body. Genome-wide analyses of the three horse populations showed that all the breeds had somewhat a low level of inbreeding within each population. In the population structure analysis, we found that Jeju crossbred was genetically closer to Thoroughbred than Jeju horse. Furthermore, we identified several signatures of selection which might be associated with traits of interest. To our current knowledge, this study is the first genomic research, analyzing genetic relationships of Jeju horse, Thoroughbred and Jeju crossbred.

Differential gene-expression profiles from canine cumulus cells of ovulated versus in vitro-matured oocytes.

We compared the nuclear maturation status and gene-expression profiles of canine cumulus cells (CCs) derived from cumulus-oocyte complexes (COCs) that were spontaneously ovulated versus those that were matured in vitro. Cumulus-oocyte complexes were retrieved from uteri by surgical flushing (after spontaneous ovulation) or by ovariectomy follicle aspiration and in vitro maturation. The objective of Experiment 1 was to investigate the nuclear maturation status of in vivo- versus in vitro-matured oocytes. The objective of Experiment 2 was to compare gene-expression profiles of CCs derived from in vivo- versus in vitro-matured COCs. Genes analysed are related to cell maturation, development and apoptosis, including GDF9, MAPK1, PTX3, CX43, Bcl2 and BAX; mRNA expression for all of these genes, except for GDF9, differed (P<0.05) between in vivo- and in vitro-matured CCs. In conclusion, we found that gene-expression profiles are related to the quality of CCs and therefore posit that monitoring gene expression could be a useful strategy to guide attempts to improve in vitro culture systems.

Effect of feeding fermented rice punch on oestrous induction in anoestrous bitches.

INTRODUCTION: As a consequence of poor productivity caused by a long anoestrous period, considerable research effort has been given to oestrus induction in dogs to enhance the productivity of young dogs and to preserve breeds. MATERIALS AND METHODS: Oestrus was induced in 30 anoestrous bitches more than three months after the last oestrus. Bitches orally received fermented rice punch with or without bromocriptine once daily for 21 consecutive days. The bitches were divided into two groups (n=10 per group): Group (1) fed fermented rice punch and Group (2) administered bromocriptine (100 µg/kg/day) and fed fermented rice punch. RESULTS: The concentration of dopamine in fermented rice punch was 47.2 mg/kg (parts per million). Six of 10 (60.0 per cent) and seven of 10 (70.0 per cent) bitches showed pro-oestrual bleeding in Groups 1 and 2, respectively. The mean and median values (min-max) to oestrus induction was not significantly different between Groups 1 and 2 (9.7±7.3, 6.5 (3-22) and 11.3±6.6, 7.9 (5-21) days) after treatment commencement (P>0.05). The pregnancy rate was very similar between Groups 1, 2 (66.0%) and control (66.0, 57.0 and 50.0 per cent). The mean and median values (min-max) of pups per bitch are also not significantly different between Groups 1, 2 and control (7.0±1.8, 7.0 (5-9) and 7.5±2.1, 7.5 (5-10) and 7.0±0, 7.0 (7-7)). CONCLUSION: We suggest that rice punch effectively induces oestrus in bitches.

Cell-free extract from porcine induced pluripotent stem cells can affect porcine somatic cell nuclear reprogramming.

Pretreatment of somatic cells with undifferentiated cell extracts, such as embryonic stem cells and mammalian oocytes, is an attractive alternative method for reprogramming control. The properties of induced pluripotent stem cells (iPSCs) are similar to those of embryonic stem cells; however, no studies have reported somatic cell nuclear reprogramming using iPSC extracts. Therefore, this study aimed to evaluate the effects of porcine iPSC extracts treatment on porcine ear fibroblasts and early development of porcine cloned embryos produced from porcine ear skin fibroblasts pretreated with the porcine iPSC extracts. The Chariot(TM) reagent system was used to deliver the iPSC extracts into cultured porcine ear skin fibroblasts. The iPSC extracts-treated cells (iPSC-treated cells) were cultured for 3 days and used for analyzing histone modification and somatic cell nuclear transfer. Compared to the results for nontreated cells, the trimethylation status of histone H3 lysine residue 9 (H3K9) in the iPSC-treated cells significantly decreased. The expression of Jmjd2b, the H3K9 trimethylation-specific demethylase gene, significantly increased in the iPSC-treated cells; conversely, the expression of the proapoptotic genes, Bax and p53, significantly decreased. When the iPSC-treated cells were transferred into enucleated porcine oocytes, no differences were observed in blastocyst development and total cell number in blastocysts compared with the results for control cells. However, H3K9 trimethylation of pronuclear-stage-cloned embryos significantly decreased in the iPSC-treated cells. Additionally, Bax and p53 gene expression in the blastocysts was significantly lower in iPSC-treated cells than in control cells. To our knowledge, this study is the first to show that an extracts of porcine iPSCs can affect histone modification and gene expression in porcine ear skin fibroblasts and cloned embryos.

Wee1B depletion promotes nuclear maturation of canine oocytes.

Most mammalian oocytes are arrested at the germinal vesicle stage by activation of Wee1B. Meiotic resumption is regulated by inactivation of Wee1B and activation of cell division cycle 25B. The aim of this study was to determine whether treatment with Wee1B-targeting small interfering RNA (Wee1B-siRNA) promotes nuclear maturation of canine oocytes from germinal vesicle stage to metaphase II (MII) stage. In experiment 1, the percentage of canine oocytes that matured to MII stage was higher (P < 0.05) among oocytes cultured in vitro for 72 hours than among those cultured for 24 and 48 hours (5.4 ± 2.5% vs. 0.0 ± 0.0% and 1.4 ± 1.0%, respectively). Furthermore, the percentage of oocytes that matured to metaphase I (MI) stage was higher (P < 0.05) among oocytes cultured for 48 and 72 hours than among those cultured for 24 hours (14.9 ± 10.0% and 22.4 ± 8.1%, respectively, vs. 5.7 ± 6.0%). In experiment 2, canine oocytes were intracytoplasmically microinjected with Wee1B-siRNA (50 µM) at various culture time points (0, 24, 48, or 72 hours). The nuclear configuration of the exception of oocytes in the 72-hour group was examined after 84 hours of culture. The percentage of oocytes that matured to the MII stage was higher (P < 0.05) among those treated with Wee1B-siRNA at 0 hours than among control oocytes and those injected at 72 hours (18.0 ± 1.7% vs. 2.1 ± 2.8% and 0.0 ± 0.0%, respectively). Moreover, the percentage of oocytes that matured to the MI stage was higher (P < 0.05) among those injected at 0 hours than among control oocytes and those injected at 24 and 72 hours (45.9 ± 6.8% vs. 22.1 ± 3.5%, 22.8 ± 10.0%, and 10.0 ± 4.4%, respectively). In experiment 3, oocytes were intracytoplasmically microinjected with Wee1B-siRNA at 0 hours of IVM and cultured for 0, 24, 48, or 72 hours. Thereafter, maturation-related gene expression was analyzed by quantitative real-time polymerase chain reaction. Messenger RNA expression of cAMP and cell division cycle 25B was lower (P < 0.05) in oocytes injected at 48 hours than in the other groups. Messenger RNA expression of cAMP was lower (P < 0.05) in oocytes injected at 0 hours than in control oocytes and those injected at 72 hours. Messenger RNA expression of mitogen-activated protein kinase 1 and mitogen-activated protein kinase 3 was higher (P < 0.05) in oocytes injected at 72 hours than in the other groups. In conclusion, we confirmed that Wee1B-siRNA microinjection enhances the percentages of canine oocytes that reach the MI and MII stages. These data suggest that Wee1B-siRNA microinjection could be a useful strategy to obtain mature canine oocytes for research and assisted canine reproduction.

In vitro development of canine somatic cell nuclear transfer embryos in different culture media.

The objective of the present study was to investigate the effects of three different culture media on the development of canine somatic cell nuclear transfer (SCNT) embryos. Canine cloned embryos were cultured in modified synthetic oviductal fluid (mSOF), porcine zygote medium-3 (PZM-3), or G1/G2 sequential media. Our results showed that the G1/G2 media yielded significantly higher morula and blastocyst development in canine SCNT embryos (26.1% and 7.8%, respectively) compared to PZM-3 (8.5% and 0%or mSOF (2.3% and 0%) media. In conclusion, this study suggests that blastocysts can be produced more efficiently using G1/G2 media to culture canine SCNT embryos.

Deciphering the genetic blueprint behind Holstein milk proteins and production.

Holstein is known to provide higher milk yields than most other cattle breeds, and the dominant position of Holstein today is the result of various selection pressures. Holstein cattle have undergone intensive selection for milk production in recent decades, which has left genome-wide footprints of domestication. To further characterize the bovine genome, we performed whole-genome resequencing analysis of 10 Holstein and 11 Hanwoo cattle to identify regions containing genes as outliers in Holstein, including CSN1S1, CSN2, CSN3, and KIT whose products are likely involved in the yield and proteins of milk and their distinctive black-and-white markings. In addition, genes indicative of positive selection were associated with cardiovascular disease, which is related to simultaneous propagation of genetic defects, also known as inbreeding depression in Holstein.

Human extracellular superoxide dismutase (EC-SOD) expression in transgenic chicken.

Extracellular superoxide dismutase (EC-SOD) is a metalloprotein and functions as an antioxidant enzyme. In this study, we used lentiviral vectors to generate transgenic chickens that express the human EC-SOD gene. The recombinant lentiviruses were injected into the subgerminal cavity of freshly laid eggs. Subsequently, the embryos were incubated to hatch using phases II and III of the surrogate shell ex vivo culture system. Of 158 injected embryos, 16 chicks (G0) hatched and were screened for the hEC-SOD by PCR. Only 1 chick was identified as a transgenic bird containing the transgene in its germline. This founder (G0) bird was mated with wild-type hens to produce transgenic progeny, and 2 transgenic chicks (G1) were produced. In the generated transgenic hens (G2), the hEC-SOD protein was expressed in the egg white and showed antioxidant activity. These results highlight the potential of the chicken for production of biologically active proteins in egg white.

Rho GTPase activating protein 15 (arhGAP15) siRNA effect apoptosis-induced by ethanol in bovine fibroblast cells.

The Rho GTPases are the sub-group of Ras super family and identified in all eukaryotes. The Rho GTPases effect different cellular signaling pathways involved in a number of diseases such as cancer, neurological and cardiovascular disorders. Members of Rho GTPases including RhoA, RhoC and Rac1 play a major role in regulation of apoptosis in different kind of stress conditions. Here we investigated the Rho GTPase activating protein 15 (ArhGAP15) gene knock-down effect on apoptosis induced by ethanol in bovine fibroblast cells. The bovine Fibroblast cells were treated and transfected with two different concentrations (50 and 100 nM) of ArhGAP15 siRNA for 48 h respectively. Both concentrations of siRNA were effective and the results of RT-PCR revealed an efficient knock-down of ArhGAP15 mRNA in fibroblast cells. Further, the normal cells exposed to a 100 mM ethanol concentration showed a reduction in cell viability and induced the ratio of apoptosis related Bax/Bcl-2 proteins compared with ArhGAP15 siRNA transfected ethanol treated cells. Ethanol also increased caspase-3 expression in normal fibroblast cells compared with transfected cells. The ArhGAP15 knock-down cells treated with ethanol decreased Bax/Bcl-2 ratio and lower caspase-3 protein levels in ArhGAP15 knocked-down cells. Our results suggest that apoptosis induced by ethanol involves the activation of Rho GTPase activating protein 15 and silencing of the said gene protects apoptosis.

No long-term feeding toxicities on the health status in rats fed with cloned Korean native beef cattle (Hanwoo) meat.

This study was designed to undertake a risk assessment to identify the health status of rats fed with somatic cell nuclear transfer (SCNT)-cloned Korean native beef cattle (Hanwoo) meat for 26 weeks. The rats were randomly divided into 5 groups, each consisting of 12 male (142.6 ± 5.23 g) and 12 female (113.7 ± 6.31 g) rats each. The animals were fed commercial pellets (control), pellets containing 5% (N-5) and 10% (N-10) of normal cattle meat, and diets containing 5% (C-5) and 10% (C-10) of cloned cattle meat. The mortality; clinical signs; body weight; food consumption; urinary, hematology, blood biochemistry, and histopathological analyses; and absolute and relative organ weights were analyzed and compared. During the 26-week test period, health status-related factors of the rats fed on cloned Hanwoo meat were found to have no test substance-related toxicities. The only difference was the increased uterus weight in female C-10 rats as compared to their counterparts counterparts (p < .05). On the basis of these health status results, it can be postulated that no food consumption risks might arise from the long-term feeding of cloned cattle meat in rats.

Quantitative analysis of sperm mRNA in the pig: relationship with early embryo development and capacitation.

Although it is well known that mRNA is present in mammalian spermatozoa, the relevance of mRNA to capacitation and early embryo development in the pig remains unclear. In the present study, we investigated differences in the abundance of selected mRNAs coding for MYC, CYP19, ADAM2, PRM1 and PRM2 in purified porcine spermatozoa depending on embryo cleavage rate and capacitation (n=20 semen samples). Semen samples were used in IVF procedures, with subsequent embryo development classified into one of two groups based on cleavage rate (i.e. high (>75%) and low (<75%) cleavage groups) and mRNA abundance in purified spermatozoa compared between these two groups. In addition, mRNA abundance was compared between capacitated and non-capacitated spermatozoa. Comparison of mRNA levels between porcine spermatozoa revealed that the abundance of MYC, CYP19, ADAM2, PRM1 and PRM2 mRNA was significantly greater in the high cleavage group (n=10 high cleavage group semen samples) than in the low cleavage group (n=10; P<0.05). Significant downregulation of MYC mRNA was observed in capacitated spermatozoa (n=12; P<0.05). The results of the present study suggest that the amount of specific mRNAs could be used for estimating the quality of spermatozoa in the pig.

Antiapoptotic effects of Phe140Asn, a novel human granulocyte colony-stimulating factor mutant in H9c2 rat cardiomyocytes.

Granulocyte colony-stimulating factor (G-CSF) is used for heart failure therapy and promotes myocardial regeneration by inducing mobilization of bone marrow stem cells to the injured heart after myocardial infarction; however, this treatment has one weakness in that its biological effect is transient. In our previous report, we generated 5 mutants harboring N-linked glycosylation to improve its antiapoptotic activities. Among them, one mutant (Phe140Asn) had higher cell viability than wild-type hG-CSF in rat cardiomyocytes, even after treatment with an apoptotic agent (H2O2). Cells treated with this mutant significantly upregulated the antiapoptotic proteins, and experienced reductions in caspase 3 activity and PARP cleavage. Moreover, the total number of apoptotic cells was dramatically lower in cultures treated with mutant hG-CSF. Taken together, these results suggest that the addition of an N-linked glycosylation was successful in improving the antiapoptotic activity of hG-CSF, and that this mutated product will be a feasible therapy for patients who have experienced heart failure.

Proteins associated with reproductive disorders in testes of human erythropoietin gene-harboring transgenic boars.

To investigate reproductive disorder in human erythropoietin (EPO)-expressing pig, we performed comparative proteomic analyses of testicular tissues from human erythropoietin (hEPO) gene-harboring transgenic pigs and wild type pigs born from natural conception. In hEPO TG pigs, we found relatively low sperm motility and higher death rate indicating impaired sperm development. Consistently, plasma concentration of testosterone was significantly lower in the transgenic post-pubertal boars compared with wild type boars. Normalized protein spots showing higher than 2-fold differential expression intensity in two-dimensional polyacrylamide gel electrophoresis were selected for matrix associated laser desorption/ionization time-to-flight mass spectrometry analysis. Specific proteins were identified by searching the NCBI protein sequence databases. Among 55 proteins selected, 12 proteins were identified as those differentially expressed between transgenic and wild type pigs. Three downregulated proteins (ß-globin, carbonyl reductase 1, and peroxiredoxin 6) and nine upregulated proteins (cytoskeletal ß-actin, α 2,3-sialyltransferase, apolipoprotein A-I, tubulin α-1A chain, tropomodulin 3, thioredoxin, heat shock Protein 70.2, ch4/domains of swine IgM, and albumin), all of which are closely related to apoptosis and cytoskeletal development, were found in the transgenic boar testes. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay confirmed the increased occurrence of apoptosis in the transgenic boar testes compared with the wild type boar testes. Reproductive defects of the hEPO-expressing transgenic pigs may be caused by the abnormal expression of the genes identified in this study.

Phenotypic characterization of Hanwoo (native Korean cattle) cloned from somatic cells of a single adult.

We investigated phenotypic differences in Hanwoo cattle cloned from somatic cells of a single adult. Ten genetically identical Hanwoo were generated by somatic cell nuclear transfer from a single adult. Weights at birth, growing pattern, horn and noseprint patterns were characterized to investigate phenotypic differences. The weights of clones at 6 and 12 months were slightly heavier than that of the donor. A horn pattern analysis revealed that seven clones had exactly the same horn pattern as the donor cow, whereas three were different. Although similarities such as general appearance can often be used to identify individual cloned animals, no study has characterized noseprint patterns for this end. A noseprint pattern analysis of all surviving clones showed that all eight animals had distinct noseprints. Four were similar to the donor, and the remaining four had more secondary-like characteristics.

Modulation by the GABA(B) receptor siRNA of ethanol-mediated PKA-α, CaMKII, and p-CREB intracellular signaling in prenatal rat hippocampal neurons.

Fetal alcohol syndrome (FAS) is a developmental neuropathology resulting from in utero exposure to ethanol; many of ethanol's effects are likely to be mediated by the neurotransmitter γ-aminobutyric acid (GABA). We studied modulation of the neurotransmitter receptor GABA(B)R and its capacity for intracellular signal transduction under conditions of ethanol treatment (ET) and RNA interference to investigate a potential role for GABA signaling in FAS. ET increased GABA(B1)R protein levels, but decreased protein kinase A-α (PKA-α), calcium/calmodulin-dependent protein kinase II (CaMKII) and phosphorylation of cAMP-response element binding protein (p-CREB), in cultured hippocampal neurons harvested at gestation day 17.5. To elucidate GABA(B1)R response to ethanol, we observed the effects of a GABA(B)R agonist and antagonist in pharmacotherapy for ethanol abuse. Baclofen increased GABA(B)R, CaMKII and p-CREB levels, whereas phaclofen decreased GABA(B)R, CaMKII and p-CREB levels except PKA-α. Furthermore, when GABA(B1)R was knocked down by siRNA treatment, CaMKII and p-CREB levels were reduced upon ET. We speculate that stimulation of GABA(B1)R activity by ET can modulate CaMKII and p-CREB signaling to detrimental effect on fetal brain development.

Enhanced biological effects of Phe140Asn, a novel human granulocyte colony-stimulating factor mutant, on HL60 cells.

Granulocyte colony-stimulating factor (G-CSF) is a cytokine secreted by stromal cells and plays a role in the differentiation of bone marrow stem cells and proliferation of neutrophils. Therefore, G-CSF is widely used to reduce the risk of serious infection in immunocompromised patients; however, its use in such patients is limited because of its non-persistent biological activity. We created an N-linked glycosylated form of this cytokine, hG-CSF (Phe140Asn), to assess its biological activity in the promyelocyte cell line HL60. Enhanced biological effects were identified by analyzing the JAK2/STAT3/survivin pathway in HL60 cells. In addition, mutant hG-CSF (Phe140Asn) was observed to have enhanced chemoattractant effects and improved differentiation efficiency in HL60 cells. These results suggest that the addition of N-linked glycosylation was successful in improving the biological activity of hG-CSF. Furthermore, the mutated product appears to be a feasible therapy for patients with neutropenia.