A simplified genetic design for mammalian enamel

A biomimetic replacement for tooth enamel is urgently needed because dental caries is the most prevalent infectious disease to affect man. Here, design specifications for an enamel replacement material inspired by Nature are deployed for testing in an animal model. Using genetic engineering we created a simplified enamel protein matrix precursor where only one, rather than dozens of amelogenin isoforms, contributed to enamel formation. Enamel function and architecture were unaltered, but the balance between the competing materials properties of hardness and toughness was modulated. While the other amelogenin isoforms make a modest contribution to optimal biomechanical design, the enamel made with only one amelogenin isoform served as a functional substitute. Where enamel has been lost to caries or trauma a suitable biomimetic replacement material could be fabricated using only one amelogenin isoform, thereby simplifying the protein matrix parameters by one order of magnitude.     

稳定表达狂犬病病毒CTN-1V株糖蛋白细胞系的建立

目的构建能稳定表达狂犬病病毒糖蛋白的CHO细胞系。方法用RT—PCR法扩增和分离CTN-1V株狂犬病毒糖蛋白基因,测序后克隆入pCDNA5．0FRT载体,构建重组质粒pCDNA5．0FRT—G,之后与POG44质粒共转染CHO细胞,采用潮霉素B抗性筛选,挑选阳性克隆,间接免疫荧光和WesternBlot进行稳定表达细胞系的鉴定。结果经酶切和测序鉴定,获得重组表达质粒pCDNA5．0FRT—G,共转染CHO细胞后,获得肉眼可见阳性细胞克隆,刮取阳性克隆进行传代扩大培养并定义为第2代,经过10代后免疫荧光和WesternBlot结果依然阳性。结论成功建立稳定表达狂犬病病毒糖蛋白的CHO细胞系,为深入研究糖蛋白的功能奠定基础。     

Genetic instability of in vitro cell lines: Implications for genetic toxicity testing

Cell line-based in vitro testing has been widely used as an important component of the genotoxicity testing battery; however, the use of cell lines is constrained by several limitations, including the genetic drift and variability. A study recently reported in the literature comprehensively examined genomic changes in a large number of cell lines and reported extensive genetic variations within the same cell lines across passage numbers and laboratories, even for single-cell derived subclones. The primary objective of this communication is to raise awareness and stimulate discussion within the genotoxicity testing community of the extent of genetic variability of cell lines in general and how these variables could potentially influence the results and reproducibility of genotoxicity testing. Meanwhile, some recommendations for good cell culture practices are highlighted to mitigate, at least to some extent, the concern about genetic variation. Environ. Mol. Mutagen. 60:559–562, 2019. © 2019 Wiley Periodicals, Inc.     

Experimental design and statistical analysis considerations for in vitro mammalian cell transformation assays with BALB/3T3 cells

Many mammalian cell assays testing for mutagenic activity have common features which cause statistical estimation and analysis problems. Such assays measure the number of cell alterations occurring in a plate containing an unknown number of cells at risk. The number of cells at risk can be estimated from a parallel cytotoxicity study. While the Poisson distribution has been assumed to apply to standardized frequencies, this is questioned. The failure of standardized frequencies to follow a Poisson distribution is attributed to the relatively small and dosage-dependent number of susceptible cells per plate. A minimum number of such cells per plate or random cluster of plates has been determined for each dose so that the measured variable approximates a Poisson distribution. A transformation is suggested to achieve reasonable normality and variance equality, thereby allowing the use of parametric analysis of variance and regression methods and an estimation of required sample sizes.     

乙型肝炎病毒表面抗原突变体稳定表达细胞系的建立和抗原性鉴定

目的 探讨乙型肝炎(乙肝)病毒(HBV)表面抗原(HBsAg)阴性HBV感染的分子机制。方法 在对46例HBsAg阴性但血清HBVDNA阳性感染者的S基因序列进行分析的基础上，构建了6株新的HBsAg变异株(4株联合点突变株，2株插入变异株)的EBO-plpp真核表达载体，并转染了COS7细胞，建立了这6株HBsAg变异株的稳定表达细胞系。分别用酶联免疫法(ELISA)和放射免疫法(RIA)对细胞表达的HBsAg抗原性进行检测。结果 除1例插入变异株可检测到较弱的阳性外，其余5株均检测不到HBsAg的存在。结论 新发现的HBsAg联合点突变和氨基酸插入变异，均对HBsAg的抗原性有负面的影响，是造成HBsAg阴性HBV感染的直接原因。     

Fingerprinting cell lines: Use of human hypervariable DNA probes to characterize mammalian cell cultures

Hypervariable DNA sequences may be used as probes to derive DNA fingerprints for individuals. To assess the use of the human 33.15 and 33.6 probes (isolated by Jeffreys and coworkers) for characterizing cell lines of nonhuman origin, DNA from different stocks of Chinese hamster (CH) cells was screened. All CHO (ovary) sublines could be readily distinguished from CH-V79 sublines by their fingerprints, but where two stocks had been derived recently from the same line, their fingerprints were nearly identical. Similarly fingerprints of HPRT-deficient mutants derived from one cell stock were identical. A V79 × CHO fusion hybrid showed equal fingerprint band-sharing with each parent line, while early-passage diploid CH cells had a fingerprint closer to CHO than to V79. Thus these data introduce a simple means of typing cell lines to avoid cross-contamination, of checking cell hybrids, and of assessing the divergence of cell stocks from one another.     

Regulation of T cells in asthma: implications for genetic manipulation

PURPOSE OF THE REVIEW: Allergic asthma is a disease characterized by airway hyperresponsiveness, inflammation and remodeling. In the past few decades it has become clear that the pathogenesis and development of this disease is controlled by cytokines released by CD4 T helper type 2 lymphocytes that develop under the influence of natural killer lymphocytes. At birth, T cell priming exhibits a T helper type 2 bias and the development of the T helper phenotype is determined in the first year of life by environmental exposure to virus or bacterial substances or environmental allergens in genetically predisposed individuals. Decreased exposure to infection in early childhood has thus been linked to the increased incidence of asthma in industrialized countries (hygiene hypothesis). In this review, we discuss the possibility that the kind and the quantity of infectious agent determines the type of immune response. RECENT FINDINGS: It has previously been shown that Toll-like receptors are involved in the recognition of intermediate components, which is the result of processed foreign antigens or damage products (produced during infection, damage or inflammation). In addition, the protective effect against allergic diseases is mediated by a new subset of CD4 T cells: the T-regulatory cells. SUMMARY: The kind and dose of antigen or infectious agent determines the development of a T helper type 1 or type 2 immune response and the activation of T-regulatory cells. The latter are known to play an important role in downregulating allergic immune responses.     

Virus-like particles occurring in cultures of stable pig kidney cell lines

Summary Virus-like particles resembling those seen in baby hamster kidney (BHK-21) cell cultures and in mouse leukemia have been found in two stable pig kidney cell lines, PK-13 and IB-RS-2. The particle's average outer diameter is c. 90 m, while the core is c. 60 m.     

Electroporation as a technique for the transfer of macromolecules into mammalian cell lines

Summary Numerous methods have been devised to facilitate the introduction of exogenous compounds into cells. The technique of electroporation allows the direct physical transfer of numerous kinds of molecules into essentially any cell, but the major application has been for transfection of DNA and this emphasis is recapitulated here. However, the conditions for transfer of DNA or other macromolecules are sufficiently similar that the same protocol is followed regardless. In addition, as electroporation involves a mechanism distinct from that of most other methods of transfection, it has distinct advantages and disadvantages relative to other transfection techniques. This review is designed to allow one to simplify the processes of determining whether electroporation is appropriate to a given experimental design, to indicate how to minimize the disadvantages, and to simplify the requisite process of parameter optimization required to evaluate and apply electroporation to the system of choice. Practical aspects are highlighted but the theoretical bases are discussed when relevant for application of the technique.     

Expression and characterization of equine herpesvirus 1 glycoprotein D in mammalian cell lines

Summary Equine herpesvirus 1 glycoprotein D (EHV-1 gD) expressed constitutively in mammalian cell lines had similar electrophoretic mobility to gD produced in EHV-1 infected cells but lacked a possibly complexed higher molecular weight form seen in the latter. Recombinant gD was N-terminally cleaved at the same site as gD in EHV-1 infected cells and expression was associated with enhanced levels of cell-cell fusion, indicating a role for EHV-1 gD in cell-to-cell transmission of virus.     

Mitotic segregation of cytoplasmic determinants for chloramphenicol resistance in mammalian cells I: Fusions with mouse cell lines

The segregation of cytoplasmically inherited chloramphenicol (CAP) resistance in mouse cells was investigated in fusions between CAP-resistant cells or cytoplasts (enucleated cells) and CAP-sensitive cells of varying tissue origin. All hybrids formed in cell-cell fusions were initially CAP-resistant, indicating that CAP resistance is dominant. Hybrids from fusions of cells of the same tissue origin (homologous) were stably CAP-resistant, whereas the hybrid population from fusions of different origins (heterologous) showed a rapid diminution of average CAP resistance. Individual hybrid clones from these heterologous fusions also showed an overall loss of CAP resistance, and a wide variation in CAP resistance which is consistent with a large number of genetic determinants (possibly mitochondrial DNA molecules) contributing to the CAP phenotype. Similar results were obtained from cytoplast-cell fusions, so the observed CAP segregation is not the result of nuclear-nuclear interactions. This segregation of CAP resistance constitutes a second criterion of cytoplasmic inheritance in mammalian cells.     

Mitotic segregation of cytoplasmic determinants for chloramphenicol resistance in mammalian cells II: Fusions with human cell lines

Cytoplasmically inherited chloramphenicol (CAP) resistance in human cells has been used to study the interaction between sensitive and resistant mitochondria. Cybrids between two HeLa cells were stable for resistance, grew rapidly and cloned well in CAP, and were O₂ tolerant. HeLa-HeLa hybrids were also stable up to 70 doublings in the absence of CAP. Cybrids between HeLa and WI-L2 cells were unstable for resistance for up to 40 doublings, grew slowly and cloned poorly in CAP, and were O₂ sensitive (S phase). The growth rate then increased and the cells became stable for resistance, cloned well, and were not O₂ sensitive (F phase). Doubling time for S but not F phase cells was proportional to CAP concentration, indicating that both kinds of mitochondria were present and functioning. The instability of CAP resistance in many interstrain but not in intrastrain mouse and human cybrids and hybrids is interpreted in relation to lower eukaryotes.     

Applications of gene transfer to study rna splicing in mammalian cell lines

Summary The first methods available for studying the signals required for splicing of eukaryotic pre-mRNA involved the transfection of cloned genes into cells in culture. The method is still of considerable importance for investigating splice site selection and for identifying the signals that modulate alternative patterns of splicing during differentiation. A method is described for the transfection of myoblast cell lines. The techniques available for preparation of RNA from transfected cells are reviewed and procedures are given for the analysis of splice site use by mapping with nuclease S1 or RT-PCR.     

A method for the isolation of purine nucleoside phosphorylase-deficient variants of mammalian cell lines

A method is described for the isolation of purine nucleoside phosphorylase (PNP) deficient lines of both Novikoff hepatoma and CHO cells. This method utilizes the ability of these cell lines to grow on inosine as a carbon and energy source. Using BrdU-light selection, inosine-negative survivors appeared at a frequency of about 10^–5 and PNP-deficient cells appeared at a frequency of about 10^–6. The PNP-negative cell line isolated from Novikoff hepatoma cells, ino-6, was temperature sensitive for both growth and PNP activity. The low activity seen after growth at 37° was approximately 5% of the wild-type level and had a tenfold increase in the apparent K_m for inosine. The extracts of the variant contained a protein that cross-reacted with antiserum made against purified rat liver PNP. This method should be useful for the isolation of PNP-deficient cells in any cell line capable of utilizing inosine for growth.     

Distinct interferon response in bat and other mammalian cell lines infected with Pteropine orthoreovirus

Virus Genes - Bats serve as natural hosts of Pteropine orthoreovirus (PRV), an emerging group of bat-borne, zoonotic viruses. Bats appear to possess unique innate immune system responses that can...