Development and characterization of a continuous cell line PSCF from Puntius sophore

A continuous Puntius sophore caudal-fin (PSCF) cell line of the pool barb Puntius sophore, an important freshwater food and ornamental fish of Asia and South East Asia, was developed from the caudal fin for the first time. The cell line was optimally maintained at 28° C in Leibovitz-15 (L-15) medium supplemented with 10% foetal bovine serum (FBS). The cytogenetic analysis revealed a diploid count of 50 chromosomes at 25th and 50th passage and 52 chromosomes at passage 70, 85 and 100. The viability of the PSCF cell lines was 75% after 6 months of storage in liquid nitrogen (-196° C). The most striking feature of the PSCF cells was its high increased growth ratio as evident from the population doubling time of 25 h at passage 100. The origin of the cell lines was confirmed by the amplification of 581 and 655 bp fragments of 16 S rRNA and cytochrome oxidase subunit I (COI) of mitochondrial DNA (mtDNA) genes, respectively. The PSCF cells were successfully transfected with green fluorescent protein (GFP) reporter plasmids and the expression of GFP gene in the cells indicated the potential utility of the cells in gene expression studies.     

Establishment and characterization of two fetal fibroblast cell lines from the yak

The objective of this work was to not only establish two fetal fibroblast cell lines from yak lung and ear tissue using a primary explant technique and cell cryogenic preservation technology but also check for their quality and biological characteristics. The cells showed typical morphologic characteristics of fibrous and long spindle appearance. Outgrowth of fibroblast-like cells from the lung and ear explants was around 2 and 3 d, and reaching 90% confluence level was in the ninth day and the thirteenth day, respectively. Biological analysis showed that the average viability of the lung fibroblast cells (ear fibroblast cells) was 97.5% (95.0%) before freezing and 91.0% (89.5%) after thawing. Analysis of the growth of the fifth passage culture revealed an ??S??-shaped growth curve with the population doubling times of 30 h for lung fibroblast cell line and 35 h for ear fibroblast cell line. Karyotyping indicated the chromosome number of yak was 2n?=?60, comprising 29 pairs of autosomes and one pair of sex chromosomes (XY). All somatic chromosomes were telocentric autosomes except that the two sex chromosomes were submetacentric. Assays for bacteria, fungi, and mycoplasmas were negative. Immunocytochemical staining showed that the cells were positive for the expression of vimentin and negative for the expression of cytokeratin. In conclusion, two yak fetal fibroblast cell lines (YFLF and YFEF) from lung and ear explants are successfully established in culture. It will not only preserve the genetic resources of yaks at the cellular level but also provide valuable materials for somatic cell cloning and transgenic research.     

The somatic chromosomes of a wild population of rats: numerical polymorphism

The karyotype and quantitative characteristics of a wild population of rats, Rattus rattus, were studied. Individuals of the population were classified into three distinct groups, each with a characteristic chromosome number of 38, 42 and ± 54 respectively. The frequency distribution of the three groups of rats in the sample studied was as follow: group I with 38 chromosomes formed 14%, group II with 42 chromosomes formed 54% and group III rats have had chromosome numbers varying between 50–60 formed 32%. The rats with 38 chromosomes had two pairs of marker chromosomes (2 long metacentric pairs). Those of group III were characterised by having a marked decrease or complete absence of short metacentric chromosomes with a simultaneous increase in the frequency of short telocentric chromosomes. Group II rats had more or less the chromosomal characteristics established for laboratory rats studied by previous workers. The total chromosomal length of somatic cells in either group I and II were found to be similar. The notable chromosomal polymorphism in number was explained in terms of centromeric fusion or dissociation.     

黑麂(Muntiacus crinifrons)细胞系的建立及其细胞生长特性和细胞遗传学分析

黑麂是我国特有的动物,其染色体大而数少(2n=8♀,2n=9♂),并且有三对大小相近,形态相似的染色体(早性6条,♂性5条)(段幸生等,1984)。由于上述特点,它可能是研究染色体结构与功能,特别是着丝点结构与功能研究的好对象,亦是辐射生物学,遗传毒理学,细胞杂交等研究的好材料。我们所建立的黑麂(♀)胚胎肺组织成纤维二倍体细胞系命名为KIZ—8102。已对其部分生物学特性进行了研究。     

Construction and behavior of circularly permuted and telocentric chromosomes in Saccharomyces cerevisiae.

We developed techniques that allow us to construct novel variants of Saccharomyces cerevisiae chromosomes. These modified chromosomes have precisely determined structures. A metacentric derivative of chromosome III which lacks the telomere-associated X and Y' elements, which are found at the telomeres of most yeast chromosomes, behaves normally in both mitosis and meiosis. We made a circularly permuted telocentric version of yeast chromosome III whose closest telomere was 33 kilobases from the centromere. This telocentric chromosome was lost at a frequency of 1.6 X 10(-5) per cell compared with a frequency of 4.0 X 10(-6) for the natural metacentric version of chromosome III. An extremely telocentric chromosome whose closet telomere was only 3.5 kilobases from the centromere was lost at a frequency of 6.0 X 10(-5). The mitotic stability of telocentric chromosomes shows that the very high frequency of nondisjunction observed for short linear artificial chromosomes is not due to inadequate centromere-telomere separation.     

Establishment and characterization of a human colonic cancer cell line expressing carbohydrate antigen 19-9 and carcinoembryonic antigen]

Colonic cancer cell strain KE43 was established from a human colonic cancer diagnosed histologically as a predominantly well differentiated adenocarcinoma with minute foci of poorly differentiated adenocarcinoma. The well differentiated adenocarcinoma cell line was identified as the major morphological picture in xenografts of KE43 and 58 in nude mice, but this changed to poorly differentiated adenocarcinoma in passage 105. Doubling time of this cancer cell line was 22.5 hours in passage 105. The modal numbers of chromosomes were 41 and 76. Cancer cells could be heterotransplanted in 100% of the nude mice. The tumor cells produced and secreted CA19-9, CEA and Laminin into the spent medium. This cell line appears to provide a useful system for studying colonic cancer in vivo and in vitro.     

藏羚羊成纤维细胞系的建立及其核型分析

藏羚羊(Pantholops hodgsonii)是我国特有的国家Ⅰ级重点保护野生动物,生存在高海拔低氧环境下,具有高原生物的细胞遗传特性。实验采用组织块贴壁培养法进行原代培养,后经差异贴壁法联合消化排除法纯化,成功构建了藏羚羊成纤维细胞系,并对培养不同代次细胞的形态、生长动力学、染色体核型等进行了研究。结果表明,培养得到的藏羚羊细胞为典型的成纤维细胞,第7、10、15代细胞群体倍增时间分别为26、48、50 h。细胞的生长均为"S"型。染色体中二倍体染色体(2n=60)占主体,所占比例为65%～91%,包括性染色体X、Y在内均为端着丝粒类型,染色体组的总相对长度为193.45,平均相对长度为3.22,未发现随体、次缢痕等特殊标志性特征。     

Sequential chromosome analysis of Walker Carcinosarcoma-256 implanted into rats after frozen storage

The Walker Carcinosarcoma-256 was preserved for 404 days at liquid nitrogen temperature. The cytological analysis of frozen cells was performed sequentially. The modal chromosome number was constantly 57 in each frozen generation. The karyotype constitutions were also stable throughout the experiments. The 6 marker chromosomes, i.e., largest submetacentric, large metacentric, large submetacentric, medium-sized metacentric, medium-sized submetacentric, and small satellited telocentric were always confirmed in each of the frozen cells analyzed. No differences existed in the results of the chromosomal observations of the frozen cells and the stock cells of the continuous animal-passaged line. The frozen-thawed specimens of cell suspension resulted in as high a percentage of successful transplants into rats as small pieces of tumor tissue. It was clearly demonstrated that tumor cells of solid Walker rat tumor, when placed in suspension, can be stored at liquid nitrogen temperatures for a year or more without chromosomal abnormality.     

A karyotype study of the cells of the African green monkey kidney cell line 4647 cultured long term in media with different sera

The karyological analysis of the cell line 4647 used for production of a killed vaccine to Hepatitis A virus was run in the 98th, 107th, 117th and 127th passages by the routine and C, G, and Ag methods of differential chromosome staining. A considerable balancing of the chromosome composition at 107-127 passage levels is shown. The cells of line 4647 present a significant heterogeneity, as to the number of chromosomes, and do not belong to any distinct modal class. The modal number of chromosomes ranged from 61 to 66 and from 121 to 125 for hyperdiploid and polyploid cells, respectively. The stable modal class of cells was established in the tetrasomic region, when culturing in the medium containing 10% CS from the 107th passage, and in the medium containing 10% FBS from the 117th passage, which conforms to one of the WHO requirements asserted to the substrate cells.     

A new continuous cell line from larval ovaries of silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

A new continuous cell line from ovarian tissue of commercial variety “Kolar Gold” of silkworm, Bombyx mori, was established and designated as DZNU-Bm-12. The tissue was grown in MGM-448 insect cell culture medium supplemented with 10% fetal bovine serum (FBS) and 3% heat-inactivated B. mori hemolymph at 25 ± 1°C. The migration of partially attached small round refractive cells from the fragments of ovarioles began from the beginning of explantation. The cells multiplied partially attached in the primary culture initially, and some of them become freely suspended after 20 passages. The cells were adapted to MGM-448 and TNM-FH media each with 10% FBS and the population doubling time of cell line was about 36 and 24 hr, respectively. The chromosome number was near diploid at initial passages and slightly increased at 176th passage, but a few tetraploids and hexaploids were also observed. DNA profiles using simple sequence repeat loci established the differences between DZNU-Bm-12 and DZNU-Bm-1 and most widely used Bm-5 and BmN cell lines. The cell line was found susceptible to B. mori nucleopolyhedrovirus (BmNPV) with 85–90% of the cells harboring BmNPV and having an average of 3–17 OBs/infected cell. We suggest the usefulness of this cell line in BmNPV-based baculoviral expression system and also for studying in vitro virus replication.     

A new cell line from the embryonic tissue of Helicoverpa armigera HBN. (Lepidoptera: Noctuidae)

A new cell line from the embryonic tissue of Helicoverpa armigera was established and designated as NIV-HA-197. It was maintained in TNM-FH medium supplemented with 10% fetal bovine serum. The cell line at passage 20 had a heterogeneous population of cells consisting of mainly epithelial-like cells (70%), followed by fibroblast-like (27%), and multinucleated giant (3%) cells. The chromosome number ranged from 45 to 185. The growth curve at passage 40 showed a fivefold increase in cell number with a population-doubling time of approximately 60 h. The cell line was found infected with the microsporidium Nosema heliothids at passage 9. Using the antiprotozoan drug Metrogyl 400 and simultaneous heat treatment, the parasite was removed from the culture. The cell line can be cryopreserved for 30 mo. The species specificity of the new cell line was determined by studying the isoenzyme profile of four enzymes, viz., lactate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, and glucose 6-phosphate dehydrogenase, and by heteroduplex analysis. Heteroduplex analysis was used to analyze the mitochondrial 16S ribosomal ribonucleic acid gene sequences along with the host insect gene sequences, and 100% homology was obtained, confirming the conspecificity of the cell line. The cell line was found to be susceptible to the baculoviruses Autographa californica multiple nucleopolyhedrovirus, Spodoptera litura multiple nucleopolyhedrovirus, and H. armigera single nucleopolyhedrovirus (HaSNPV). More than 90% of the cells were infected by HaSNPV on the seventh post infection day (PID), and 28.8 x 10(6) NPV/ml was yielded on the 10th PID. The in vitro-grown HaSNPV caused 100% mortality, when fed to the second instar H. armigera larvae, in 6 d. Cessation of feeding was observed on the second PID.     

Human retinal pigment cell culture

Human retinal pigment epithelium (RPE)-derived cell lines were established from RPE-covered choroid tissue fragments, which had been generated by culture on nontissue culture plastic. Two phenotypes were apparent in a given line: (a) a compact cell which formed domes and ultimately melanosomes before being sloughed; and (b) a squamous cell which was often elongated and which bound antibody to human keratins. This latter cell did not become black or form domes. The average number of cell doublings for the 13 lines tested was between 15 and 40 when cultured in a modified Eagle's minimum essential medium containing 10% fetal bovine serum. Cell lines newly established from material that had been in culture for more than 6 months had normal mitotic chromosomes and still developed areas with strongly pigmented cells when refed. Normal human epithelial cell lines of this kind may be useful in studies of cell aging and defining change associated with the development of neural cells from ectoderm.     

A Mouse Hepatoma Cell Line which Secretes Several Serum Proteins Including Albumin and α-foetoprotein

A permanent cell line (BW) was established from a transplantable mouse hepatoma, BW7756, which produces α-foetoprotein (AFP).Three clones were isolated from the uncloned culture: BW1, BW2 and B WTG3. The cells of the latter clone, which was isolated after selection in the presence of thioguanine, are deficient in the enzyme hypoxanthine-guanine-phosphoribosyl transferase. Both B W1 and BWTG3 cells have mean chromosome number of 64 (60 telocentric and 4 metacentric chromosomes). All three clones secrete at least'five serum proteins into the culture medium: albumin, AFP, an a2 globulin, transferrin and C3, the third component of complement. The approximate rate of albumin secretion by BW1 and BWTG3 cells is 10 μg/24 h/10⁶ cells. Both albumin and AFP can easily be detected in cell extracts. The simultaneous production of AFP and a hepatocyte specific marker (albumin) by cloned hepatoma cells show that the production of AFP by the tumour is due to the tumoural hepatocytes themselves.     

Microinjection of Intact 200- to 500-kb Fragments of YAC DNA into Mammalian Cells

DNA of yeast artificial chromosomes (YACs) was prepared for microinjection by separation from most of the natural yeast chromosomes on a pulsed-field gel, treatment with agarase, and centrifugation. A salt concentration of 100 mM NaCl was necessary to protect the DNA from shear during these procedures. Injection of a 590-kb YAC, yGART2, into Chinese hamster ovary cells gave rise to cells expressing the 40-kb human GART gene carried on the YAC. Nine of 12 cell lines analyzed contained an intact stretch of at least 110 kb of YAC DNA surrounding the GART gene, and one cell line contained at least 480 kb, but not the entire 590 kb, intact. Mouse L A-9 cells were similarly injected with DNA of a 230-kb YAC containing the human β-globin gene cluster and a mammalian selectable marker. Seven of 10 of the resulting cell lines contained both YAC vector arms plus the intact 140-kb SfiI fragment spanning the β-globin gene. Three cell lines were analyzed by Rec A-assisted restriction endonuclease (RARE) cleavage and found to contain the entire intact 210-kb YAC insert. Introduction of similarly prepared DNA into mammalian cells by lipofection gave rise to cell lines with multiple YAC fragments that were generally shorter than the YAC fragments found in microinjected cell lines. The results show that microinjection of gel-purified YAC DNA into mammalian cells is an efficient method of transferring DNA fragments several hundred kilobase pairs in size into mammalian cells.     

小鼠胚胎干细胞(ES-8501细胞)建系过程的核型及特性分析

小鼠胚胎性癌(EC)细胞系的细胞核型大多数异常,对用于分析EC细胞与胚胎细胞之间的关系和进行嵌合体研究等都是不利的。人们都期望能有正常核型的胚胎细胞系的建立。近年来Evans和Kaufman以及Martin等人先后用不同方法直接从小鼠的内细胞团(ICM)细胞建立了多潜能的胚胎干细胞(erabryonicstem eells,简称ES细胞),也有人称之为EK     

内蒙古西伯利亚棘球绦虫和多房棘球绦虫泡状蚴在小白鼠发育成熟的比较

The alveolar echinococcus is one of the most dangerous worm parasites in man. Rausch and Schiller reported a new species, Echinococcus sibiricensis n. sp. from arctic fox, Alpex logopus, on St. Lawrence Island of Alaska, USA. According to the view of Vogel, the sibiricensis form is only a geographical race or subspecies of Europe Echinococcus multilocularis. So far, the two names, Echinococcus multiocularis multilocularis and Echinococcus multilocularis sibiricensis, existed in many references and text books. We have found the adults of Echinococcus sibiricensis and Echinococcus multilocularis from sand foxes, Vulpes corsac and their larval stages (alveolar echinococcus) from field voles, Microtus brandti in the Hulunbeier Pasture of Inner Mongolia, northeastern China in 1985 and 1998-1999. Two types of metacestodes with quite different styles of early development of E. sibiricensis and E. multilocularis were found from field voles and laboratory experimental white mice. As one characteristic of alveolar E. multilocularis, the capsules are produced by the exogenous budding of germinal cell layer together with cyst wall. The protoscoleces grow from germinal cells on germinal cell layer. The peduncles of early protoscoleces attached to the germinal cell layer on the inner surface of capsule wall(Plate I, Figs. 1-2). Some protoscoleces in reticular structure were linked with the inner surface of capsule wall (Plate I, Fig. 3) in livers of mice in 9.5th month postinfection. In 14th month old alveolar multilocularis, large number of mature protoscoleces in reticular structure were still linked to the inner surface of capsule wall (Plate I, Figs. 4-8). The cavities of some capsules were filled with protoscoleces in meshes of reticular structure which were also linked around with the inner surface of capsule wall (Plate I, Fig. 9). The superficial surface of livers of positive field voles and experimental mice never showed any hyperemic phenomenon. The superficial surfaces of livers and lungs of positive field voles and experimental mice infected with alveolar E. sibiricensis were highly hyperemic. The metacestodes of E. sibiricensis composed of mother cyst, undifferentiated embryonic cysts and small brood capsules. Cavities of all cysts were fully filled with germinal cell masses. Host reaction appeared to be very strong, all cysts were surrounded by thick connective tissue and dense leukocytes (Plate II, Fig. 10). All alveolar vesicles were found located in lungs tissue of experimental mice. Large germinal cell masses metastasized out from undifferentiated embryonic cysts into host lung tissue, where germinal cell masses developed into accumulation of early protoscoleces (Plate II, Figs. 11-12). Early protoscoleces of alveolar E. sibiricensis were seen earliest in mice lung tissues on 101-104th days after infection. Many small capsules in different sizes and different shapes containing mature protoscoleces and reticular structure (Plate II, Figs. 13-15) were found in lungs of mice in 9th month after infection. Only in one experimental mouse infected with alveolar E. sibiricensis in 8.5th month postinfection, both its lung and liver existed alveolar cysts; the capsules in liver were surrounded by very thick connective tissue of the host, and there were some protoscoleces in their cavities (Plate II, Figs. 16-18).     

A mouse hepatoma cell line which secretes several serum proteins including albumin and alpha-foetoprotein.

A permanent cell line (BW) was established from a transplantable mouse hepatoma, BW7756, which produces alpha-foetoprotein (AFP). Three clones were isolated from the uncloned culture: BW1, BW2 and BWTG3. The cells of the latter clone, which was isolated after selection in the presence of thioguanine, are deficient in the enzyme hypoxanthine-guanine-phosphoribosyl transferase. Both BW1 and BWTG3 cells have mean chromosome number of 64 (60 telocentric and 4 metacentric chromosomes). All three clones secrete at least five serum proteins into the culture medium: albumin, AFP, and alpha 2 globulin, transferrin and C3, the third component of complement. The approximate rate of albumin secretion by BW1 and BWTG3 cells is 10 mug/24 h/10(6) cells. Both albumin and AFP can easily be detected in cell extracts. The simultaneous production of AFP and a hepatocyte specific marker (albumin) by cloned hepatoma cells show that the production of AFP by the tumour is due to the tumoural hepatocytes themselves.     

蛾螺科三种螺的核型观察

本文介绍了蛾螺科三种螺的核型。水泡蛾螺(Buccinum pcmphigum)为2n=30,染色体的形态分类组成为16m+10sm+4st,NF=56;Plicifusus scissuratus为2n=34,染色体的形态分类组成为20m+10sm+4st,NF=64;香螺(Neptumea cumingi)为2n=60,染色体的形态分类组成为30m+22sm+8st,NF=112。 蛾螺科核型分析,显示贝类染色体数目及其形态与贝类进化有关。     

Establishment of a turbot fin cell line and its susceptibility to turbot reddish body iridovirus

A turbot, Scophthalmus maximus, fin (TF) cell line was established and susceptibility to turbot reddish body iridovirus (TRBIV) was determined in this study. Primary culture of TF cells was initiated from fin tissue pieces partially digested with trypsin, collagenase II and hyaluronidase. Digested tissue pieces were cultured at 24 °C in Leibovitz-15 medium (pH 7.2), supplemented with 20% fetal bovine serum, carboxymethyl chitosan, N-acetylglucosamine hydrochloride, basic fibroblast growth factor and epidermal growth factor. The cultured TF cells, in fibroblast shape, proliferated to 100% confluency 50 days later. A TF cell line, with a population doubling time of 45.6 h at passage 80, has been established and subcultured to passage 133. Chromosome analyses indicated that the TF cells exhibited chromosomal aneuploidy with a modal chromosome number of 44 which displayed the normal diploid karyotype of S. maximus at least up to passage 80. TRBIV susceptibility testing demonstrated that cytopathic effect and propagated viral particles were observed in TF cells after TRBIV infection. In conclusion, a continuous TRBIV susceptible TF cell line has been established successfully, and the cell line may serve as a valuable tool for studies of cell-virus interactions and has applications for different kinds of cytotechnological studies as well.