Stress mRNA metabolism in canavanine-treated chicken embryo cells.

Four major chicken stress mRNAs with apparent molecular weights of 1.2 X 10(6), 0.88 X 10(6), 0.59 X 10(6), and 0.25 X 10(6) to 0.28 X 10(6) were separated on acidic agarose-urea gels. Using cell-free translation, the coding assignments of these mRNAs were determined to be stress proteins with apparent molecular weights of 88,000, 71,000, 35,000, and 23,000. Despite high levels of translational activity in vivo and in vitro, no newly synthesized mRNA for the 23-kilodalton stress protein was detected on gels under conditions which readily allowed detection of other stress mRNAs, suggesting activation of a stored or incompletely processed mRNA. Cloned Drosophila heat shock genes were used to identify and measure changes in cellular levels of the two largest stress mRNAs. Synthesis of these mRNAs increased rapidly during the first hour of canavanine treatment and continued at a high rate for at least 7 h, with the mRNAs attaining new steady-state levels by ca. 3 h. Both of these inducible stress mRNAs had very short half-lives compared with other animal cell mRNAs. Using an approach-to-steady-state analysis, the half-lives were calculated to be 89 min for the mRNA encoding the 88-kilodalton stress protein and 46 min for the mRNA encoding the 71-kilodalton stress protein. Chicken 18S and 28S rRNA synthesis was inhibited, and actin mRNA levels measured with cloned cDNA encoding chicken beta-actin slowly declined in canavanine-treated cells.     

Establishment and characterization of chicken embryo fibroblast clone LSCC-H32

Cell line CEC-32 and clone LSCC-H32 were established from primary chicken embryo cells spontaneously but not experimentally transformed at 32 degrees C. The lines consisted of fibroblastoid and polygonal cells and had a subtetraploid karyotype of 2N = 130 to 140. The cells showed increased plating efficiency and metabolic activities as demonstrated by hexose uptake and plasminogen activator assay. The established cells produced avian lymphoid leukosis viruses of subgroups A and B. The virus released from LSCC-H32 cells induced lymphoid leukosis in inoculated chickens 18 to 22 wk post infection (PI). The cells have been carried in continuous culture for 285 passages and they appeared to grow indefinitely. They were efficiently used to propagate several animal viruses and to titrate chicken interferon.     

Myostatin gene silencing by RNA interference in chicken embryo fibroblast cells

Myostatin (MSTN), a member of transforming growth factor-β (TGF-β) superfamily, is a negative regulator of the skeletal muscle growth, and suppresses the proliferation and differentiation of myoblast cells. Dysfunction of MSTN gene either by natural mutation or genetic manipulation (knockout or knockdown) has been reported to interrupt its proper function and to increase the muscle mass in many mammalian species. RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) has become a powerful tool for gene knockdown studies. In the present study transient silencing of MSTN gene in chicken embryo fibroblast cells was evaluated using five different shRNA expression constructs. We report here up to 68% silencing of myostatin mRNA using these shRNA constructs in transiently transfected fibroblasts (p<0.05). This was, however, associated with induction of interferon responsive genes (OAS1, IFN-β) (3.7-64 folds; p<0.05). Further work on stable expression of antimyostatin shRNA with minimum interferon induction will be of immense value to increase the muscle mass in the transgenic animals.     

Specific binding of nonsupressible insulinlike activity to chicken embryo fibroblasts and to a solubilized fibroblast receptor.

¹²⁵I-labeled nonsuppressible insulinlike activity—soluble in acid/ethanol (NSILA-S) binds to cultured chicken embryo fibroblasts and to an extract obtained by treating the fibroblasts with Triton X-100. Binding to intact cells and to the cell extract is timetemperature, and pH-dependent and shows saturation kinetics. The apparent intrinsic association constant for binding of NSILA-S to intact cells is 10⁹ M^?1, the number of binding sites per cell approximately 6000. Cold NSILA-S preparations of different purity displace bound ¹²⁵I-labeled NSILA-S according to their biological potency. Moreover, cold NSILA-S displaces bound ¹²⁵I-labeled NSILA-S in concentrations in which it also stimulates thymidine incorporation into fibroblast DNA. Insulin displaces bound ¹²⁵I-labeled NSILA-S only at concentrations above 1 mU/ml. Glucagon, ACTH, human growth hormone and inactivated NSILA-S are ineffective. The displacement curves obtained with human serum are similar to those obtained in the presence of cold NSILA-S.     

Detection of nerve growth factor mRNA in the developing chicken embryo

Nerve growth factor (beta NGF) is a protein supporting sympathetic and sensory innervation in the peripheral tissues as well as cholinergic innervation in the brain. A DNA probe derived from a genomic clone coding for chicken NGF was used to study NGF mRNA levels during development. NGF mRNA was detected in the chicken embryo as early as day 3.5 of incubation. The level of NGF mRNA in total embryo increased four-fold until day 8, remained high until day 12, and subsequently decreased. No corresponding peak in NGF mRNA expression was found in heart and brain measured separately. Instead these organs showed increased NGF mRNA levels after hatching. The highest levels of NGF mRNA in the day-8 embryo were found in skin and eye (in particular cornea, but also iris, sclera-choroid and neural retina) suggesting a correlation between sensory innervation and this early peak of NGF expression.     

Size-dependent accumulation of mRNA at the leading edge of chicken embryo fibroblasts

β-actin mRNA localizes to the leading edge of a living chicken embryo fibroblast. Recently we proposed that the mRNA maintains its localization at the leading edge by utilizing the heterogeneity of cytoplasmic microstructure (Yamagishi et al., 2009 [10]). In this study, we observed the intracellular distribution of β-actin mRNA variants to elucidate the mechanism of mRNA localization at the leading edge. We found that the degree of localization correlated positively with the molecular mass of the mRNA variants. We further demonstrated that the molecular mass-dependent localization was found even with dextrans, which have no biological function. The dependency of localization on molecular mass suggested that the barrier effect caused by the physical obstruction of the cytoplasmic microstructure is one of the major factors controlling mRNA localization in motile fibroblasts.     

Isolation of a chicken thioredoxin cDNA clone. Thioredoxin mRNA is differentially expressed in normal and Rous sarcoma virus-transformed chicken embryo fibroblasts

We have isolated a cDNA clone for chicken thioredoxin by differential screening of a cDNA library. The protein product which this clone encodes is very similar to other thioredoxins, and it displays thioredoxin activity when expressed in Escherichia coli. This clone represents the first metazoan thioredoxin for which the protein or nucleic acid sequence is known. Comparison of the chicken thioredoxin protein sequence with those from bacteria and plants indicates structural features that appear to be essential for activity. Transformation of chicken embryo fibroblasts by Rous sarcoma virus elevates the level of thioredoxin mRNA whereas the level of thioredoxin mRNA in a nonproliferative tissue (brain) is much lower than in chicken embryo fibroblasts.     

Transformation of chicken embryo fibroblast cells by avian retroviruses containing the human Fyn gene and its mutated genes.

The transforming activity of the human fyn protein, p59fyn, which is a kinase of the src family, was investigated by testing the effect of recombinant avian retrovirus (Fyn virus) expressing p59fyn on chickens or cultured chicken embryo fibroblast (CEF) cells. The Fyn virus did not induce transformed foci. After several passages of the virus stock on CEF cells, however, a few foci were detected in the presence of dimethyl sulfoxide. Chickens inoculated with Fyn virus at the stage of 12-day-old embryos developed fibrosarcomas 3 to 6 weeks after hatching. The viruses obtained from these foci and from one of the tumor tissues showed high transforming activity in the presence of dimethyl sulfoxide, suggesting that these viruses carry spontaneous mutations of the fyn gene. Four fyn genes from CEF DNAs infected with transforming viruses were molecularly cloned, and their products were confirmed to possess transforming activity. DNA sequence analysis of the fyn genes showed that two of the four mutants have Thr instead of Ile at position 338 in the kinase domain. The other two mutants carry deletions of 78 and 108 base pairs, respectively, which result in complete loss of region C of SH2. The overall level of proteins containing phosphotyrosine was significantly higher in transformed cells than in normal CEF cells. Our data indicate that when expressed at high levels in a retrovirus, normal p59fyn cannot cause cellular transformation, but that mutant p59fyn with either a single amino acid substitution in the kinase domain or a deletion including region C produces a transforming protein, perhaps due to enhanced tyrosine kinase activity. This is the first observation that deletion of region C can unmask the potential transforming activity of a src family kinase.     

Screening for gene function in chicken embryo using RNAi and electroporation

In the postgenomic era the elucidation of the physiological function of genes has become the rate-limiting step in the quest to understand the development and function of living organisms. Gene functions cannot be determined by high-throughput methods but require analysis in the context of the entire organism. This is particularly true in the developing vertebrate nervous system. Because of its easy accessibility in the egg, the chicken embryo has been the model of choice for developmental in vivo studies. However, its usefulness has been hampered by a lack of methods for genetic manipulation. Here we describe an approach that could compensate for this disadvantage. By combining gene silencing by dsRNA (through RNA interference, RNAi) with in ovo electroporation, we developed an efficient method to induce loss of gene function in vivo during the development of the chicken CNS. This method opens new possibilities for studying gene function not only by gain-of-function but also by loss-of-function approaches and therefore represents a new tool for functional genomics.     

自研培养基在人用鸡胚狂犬病疫苗中的应用

目的评估自主研发培养基QS作为首选培养基用于冻干人用狂犬病疫苗(鸡胚成纤维细胞)生产过程的可行性。方法分别制备基于自主研发培养基QS和其他3种商业化培养基(X1、X2、X3)的鸡胚成纤维细胞悬液,观察和比较细胞形态和生长特性;以Flury HEP株接种鸡胚成纤维细胞(MOI=0.003),分别通过直接免疫荧光法、蛋白质印迹法(Western blotting)和酶联免疫吸附测定(enzyme-linked immunosorbent assay, ELISA)比较不同培养基条件下收获液中病毒滴度和G蛋白含量。结果在细胞浓度1×10~6个/mL条件下,4种培养基培养的鸡胚成纤维细胞在形态上均无显著区别;但是自主研发培养基QS和X2的病毒收获液的G蛋白含量在第4天时分别为0.66 IU/mL和0.63 IU/mL,高于X1的0.5 IU/mL和X3的0.3 IU/mL。在第6天时分别为0.92 IU/mL和0.88 IU/mL,高于X1的0.64 IU/mL和X3的0.52 IU/mL,说明基于自主研发培养基QS和X2的病毒收获液在G蛋白含量方面具有明显优势。结论自主研发培养基QS可以用于冻干人用狂犬病疫苗(鸡胚成纤维细胞)的生产。     

Sex determination in the chicken embryo.

The chicken embryo represents a suitable model for studying vertebrate sex determination and gonadal sex differentiation. While the basic mechanism of sex determination in birds is still unknown, gonadal morphogenesis is very similar to that in mammals, and most of the genes implicated in mammalian sex determination have avian homologues. However, in the chicken embryo, these genes show some interesting differences in structure or expression patterns to their mammalian counterparts, broadening our understanding of their functions. The novel candidate testis-determining gene in mammals, DMRT1, is also present in the chicken, and is expressed specifically in the embryonic gonads. In chicken embryos, DMRT1 is more highly expressed in the gonads and Müllerian ducts of male embryos than in those of females. Meanwhile, expression of the orphan nuclear receptor, Steroidogenic Factor 1 (SF1) is up-regulated during ovarian differentiation in the chicken embryo. This contrasts with the expression pattern of SF1 in mouse embryos, in which expression is down-regulated during female differentiation. Another orphan receptor initially implicated in mammalian sex determination, DAX1, is poorly conserved in the chicken. A chicken DAX1 homologue isolated from a urogenital ridge library lacked the unusual DNA-binding motif seen in mammals. Chicken DAX1 is autosomal, and is expressed in the embryonic gonads, showing somewhat higher expression in female compared to male gonads, as in mammals. However, expression is not down-regulated at the onset of testicular differentiation in chicken embryos, as occurs in mice. These comparative data shed light on vertebrate sex determination in general.     

cDNA cloning and mRNA expression of Transformer 2 (Tra 2) in chicken embryo

We report the cloning of a chicken Transformer 2 (Tra 2) cDNA that encodes a protein of 289 amino acids which are 97.9% identical to those of mammalian splicing factor, Tra 2. Tra 2 mRNA was expressed in chicken embryonic tissues and was observed as a band of 1.5 kb by Northern blot analysis. Whole mount in situ hybridization showed an mRNA expression of Tra 2 in telencephalon, mandible, hyoid arch, wing and leg buds as early as day 3.5 of incubation. These results suggest that the Tra 2 gene may play a role in organogenesis in the chicken embryo.     

Aminoacylation of undermethylated mammalian transfer RNA.

To study the role of 5-methylcytidine in the aminoacylation of mammalian tRNA, bulk tRNA specifically deficient in 5-methylcytidine was isolated from the livers of mice treated with 5-azacytidine (18 mg/kg) for 4 days. For comparison, more extensively altered tRNA was isolated from the livers of mice treated with DL-ethionine (100 mg/kg) plus adenine (48 mg/kg) for 3 days. The amino acid acceptor capacity of these tRNAs was determined by measuring the incorporation of one of eight different 14C-labeled amino acids or a mixture of 14C-labeled amino acids in homologous assays using a crude synthetase preparation isolated from untreated mice. The 5-methylcytidine-deficient tRNA incorporated each amino acid to the same extent as fully methylated tRNA. The tRNA from DL-ethionine-treated livers showed an overall decreased amino-acylation capacity for all amino acids tested. The 5-methylcytidine-deficient tRNA from DL-ethionine-treated mice were further characterized as substrates in homologous rate assays designed to determine the Km and V of the aminoacylation reaction using four individual 14C-labeled amino acids and a mixture of 14C-labeled amino acids. The Km and V of the reactions for all amino acids tested using 5-methylcytidine-deficient tRNA as substrate were essentially the same as for fully methylated tRNA. However, the Km and V were increased when liver tRNA from mice treated with DL-ethionine plus adenine was used as substrate in the rate reaction with [14C]lysine as label. Our results suggest that although extensively altered tRNA is a poorer substrate than control tRNA in both extent and rate of aminoacylation, 5-methylcytidine in mammalian tRNA is not involved in the recognition of the tRNA by the synthetase as measured by aminoacylation activity.