Cell Biology

The stable HBV-transfected cell lines, which based on stable integration of replication-competent HBV genome into hepatic cells, are widely used in basic research and antiviral drug evaluation against HBV. However, previous reported strategies to generate HBV-replicating cell lines, which primarily rely on random integration of exogenous DNA by plasmid transfection, are inefficient and time-consuming. We newly developed an all-in-one Sleeping Beauty transposon system (denoted pTSMP-HBV vector) for robust generation of stable HBV-replicating cell lines of different genotype. The pTSMP-HBV vector contains HBV 1.3-copy genome and dual selection markers (mCherry and puromycin resistance gene), allowing rapid enrichment of stably-transfected cells via red fluorescence-activated cell sorting and puromycin antibiotic selection. In this protocol, we described the detailed procedure for constructing the HBV-replicating stable cells and systematically evaluating HBV replication and viral protein expression profiles of these cells.

Somatic chromosomes are usually studied from the root tip cells of the plants for cytological investigations. In dioecious plant species like Coccinia grandis, it is very difficult to get meristematic root tip cells from the mature plants of the respective sex forms. In this report, young leaves of the respective sexual phenotypes were used as tissue samples for mitotic chromosome analysis. For meiotic preparation, flower buds of appropriate size were selected for chromosomal studies. Following protocols could be effectively used for routine chromosome preparations in other plant species as well.

ROS-induced DNA damage is repaired in living cells within a temporal and spatial context, and chromatin structure is critical to a consideration of DNA repair processes in situ. It’s well known that chromatin remodeling factors participate in many DNA damage repair pathways, indicating the importance of chromatin remodeling in facilitating DNA damage repair. To date, there has been no method to induce site-specific oxidative DNA damage in living cells. Therefore, it is not known whether the DNA repair mechanisms differ within active or condensed chromatin. We recently established a novel method, DTG (Damage Targeted at one Genome-site), to study DNA damage response of reactive oxygen species (ROS)-induced DNA damage in living cell at one genome loci with active or inactive transcription. For this, we integrated a tetracycline responsive elements (TRE) cassette (~90 kb) at X-chromosome in U2OS cells (Lan et al., 2010), then fused KillerRed (KR), a light-stimulated ROS-inducer which can specifically produce ROS-induced DNA damage, to a tet-repressor (tetR-KR, OFF) or a transcription activator (TA-KR, ON) (Lan et al., 2014) (Figure 1). TetR-KR or TA-KR binds to the TRE cassette and induces ROS damage under hetero- or euchromatin states, respectively. How chromatin states regulate the DNA damage response processes can be examined by using this powerful method.

A chromosome is the structure that organizes DNA and protein in cells. It is a single piece of coiled DNA containing coding and non-coding sequences. Human cells have 23 pairs of chromosomes including 22 pairs of autosomes and one pair of sex chromosome, giving a total of 46 per cell. In tumor cells, chromosomal instability has been considered to be one of the hallmarks of tumor formation. Here we use the karyotype analysis to separate the chromosomes and observe the chromosomes in tumor cells with a microscope.

This protocol is a more detailed version of previous protocols (Yang et al., 2011; Bolaños-Villegas et al., 2013) developed for the examination of meiotic chromosome spreads. Meiotic chromosome spreads are useful to determine the presence of defects in chromosome pairing and segregation. The protocol also describes how to perform fluorescent in situ hybridization experiments with a centromere probe used to label chromosomes.

This protocol is optimized for immunoFISH staining of adherent cultured mammalian cells. It combines immunofluorescence for DNA damage response factors (e.g. 53BP1) and FISH against telomeric DNA.

Fluorescence in situ hybridization (FISH) is a method that uses a fluorescently labeled DNA probe for mapping the position of a genetic element on chromosomes. A DNA probe is prepared by incorporating Cy-3 or Cy-5 labeled nucleotides into DNA by nick-translation or a random primed labeling method. This protocol was used to map genes (Sharakhova et al., 2010) and microsatellite markers (Kamali et al., 2011; Peery et al., 2011) on polytene chromosomes from ovarian nurse cells and salivary glands of malaria mosquitoes. Detailed physical genome mapping performed on polytene chromosomes has the potential to link DNA sequences to specific chromosomal structures such as heterochromatin (Sharakhova et al., 2010). This method also allows comparative cytogenetic studies (Sharakhova et al., 2011; Xia et al., 2010), and reconstruction of species phylogenies (Kamali et al., 2012).