Correction: Gaussian decomposition of high-resolution melt curve derivatives for measuring genome-editing efficiency.

[This corrects the article DOI: 10.1371/journal.pone.

Gaussian decomposition of high-resolution melt curve derivatives for measuring genome-editing efficiency.

We describe a method for measuring genome editing efficiency from in silico analysis of high-resolution melt curve data. The melt curve data derived from amplicons of genome-edited or unmodified target sites were processed to remove the background fluorescent signal emanating from free fluorophore and then corrected for temperature-dependent quenching of fluorescence of double-stranded DNA-bound fluorophore. Corrected data were normalized and numerically differentiated to obtain the first derivatives of the melt curves.

Non-Homologous End Joining and Homology Directed DNA Repair Frequency of Double-Stranded Breaks Introduced by Genome Editing Reagents.

Genome editing using transcription-activator like effector nucleases or RNA guided nucleases allows one to precisely engineer desired changes within a given target sequence. The genome editing reagents introduce double stranded breaks (DSBs) at the target site which can then undergo DNA repair by non-homologous end joining (NHEJ) or homology directed recombination (HDR) when a template DNA molecule is available. NHEJ repair results in indel mutations at the target site.

Gammaretroviral vector encoding a fluorescent marker to facilitate detection of reprogrammed human fibroblasts during iPSC generation.

Induced pluripotent stem cells (iPSCs) are becoming mainstream tools to study mechanisms of development and disease. They have a broad range of applications in understanding disease processes, in vitro testing of novel therapies, and potential utility in regenerative medicine. Although the techniques for generating iPSCs are becoming more straightforward, scientists can expend considerable resources and time to establish this technology.

Gene therapy with drug resistance genes.

A major side effect of cancer chemotherapy is myelosuppression. Expression of drug-resistance genes in hematopoietic stem cells (HSC) using gene transfer methodologies holds the promise of overcoming marrow toxicity in cancer chemotherapy. Adequate protection of marrow cells in cancer patients from myelotoxicity in this way would permit the use of escalating doses of chemotherapy for eradicating residual disease.

Cloning and expression of canine O6-methylguanine-DNA methyltransferase in target cells, using gammaretroviral and lentiviral vectors.

The human O(6)-methylguanine-DNA methyltransferase (MGMT) gene and its mutants have been used for in vivo selection of transduced hematopoietic stem cells with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) alone or in combination with O(6)-benzylguanine (BG). To allow similar in vivo selection in dogs, without the risk of inducing an immune response, we have cloned the canine MGMT drug resistance gene. Comparison of canine and human MGMT-coding regions indicates that there is about 62% amino acid identity and 78% similarity between the two MGMTs.

Evaluation of Tat-encoding bicistronic human immunodeficiency virus type 1 gene transfer vectors in primary canine bone marrow mononuclear cells.

Tat-encoding human immunodeficiency virus type 1 (HIV-1) gene transfer vectors were evaluated in primary canine bone marrow mononuclear cells. Tat vectors provided higher levels of gene expression than vectors with internal promoters. The HIV-1 vector was also more efficient than Moloney murine leukemia virus (MoMLV) vectors for transduction of canine bone marrow mononuclear cells in vitro.

Poor expression of MDR1 transgene in HeLa cells by bicistronic Moloney murine leukemia virus-based vector.

Cotransfer of a therapeutic gene together with the human MDR1 gene provides an opportunity to increase the number of transduced marrow cells, expressing the therapeutic gene, by in vivo selection for MDR1. We have used an Lg-MDR1-IRES-neo (LgMIN) retroviral vector, containing MDR1 and neo genes, separated by the EMCV IRES. Human HeLa or canine CTAC cells, transduced with GALV env pseudotyped LgMIN at an MOI of less than 0.

[Analysis of the genome compartmentalization phenomenon in normal and neoplastic cells].

Intact cell nuclei (or whole cell lysates) were immobilized on Celite and extracted gradually with gradients of NaCl, LiCl-urea and temperature. Contrary to the notion of DNA integrity and continuity within chromosomes, a heterogeneous spectrum of DNA fragments of large size was obtained, adhesion of which to the nuclear interior widely varied. Similar chromatographic patterns of DNA were observed in analysis of various origin cells both in normal animal tissues and in malignant cells (Djungarian hamster fibroblasts transformed by SV40).

Differential dissociation of chromatin digests: a novel approach revealing a hierarchy of DNA-protein interactions within chromatin domains.

We describe here a novel approach to the dissection of chromatin structure by extracting DNA fragments from digested nuclei irreversibly immobilized (via proteins) on Celite columns. Three successive gradients (NaCl, LiCl-urea, temperature) are used to release three families of DNA fragments: namely, the 'DNA adherence' classes DNA-0, DNA-I and DNA-II, respectively. This 'protein image' DNA chromatography separates DNA fragments in accordance with the tightness of their bonds with proteins in situ.

A procedure for DNA and RNA transfer to membrane filters avoiding weight-induced gel flattening.

We describe a technique of rapid (within 1-2 h) transfer of DNA and RNA from agarose gels to nitrocellulose or nylon membrane filters. It is characterized by nearly complete elimination of mechanical action on the gel (a thin layer of liquid is placed over the gel and, filtering through the gel into a stack of paper towels beneath, it transfers nucleic acids onto the filter under the gel). This "descending" transfer, as opposed to the widely used "ascending" Southern transfer, reduces the transfer time (to about 1 h) with equal or higher quality of the hybridization signal.

[Changes in gene expression in the brain of rats with Zajdela's ascitic hepatoma].

Expression of some genes in the brain of ascitic hepatoma of Zajdela bearing rats was compared with that of control animals using Northern blot hybridization technique. The differences revealed were: an increased expression of actin gene and decreased expression of hsp70 gene in the brain of tumor-bearing animals.

[Comparative analysis of subdomain fragments of chromatin].

Nuclei isolated from Djungarian hamster fibroblasts transformed by SV40 were treated with restriction endonuclease Bsp RI, fixed on Celite columns and underwent successive gradients of dissociating agents, such as NaCl, LiCl-urea, and temperature. This procedure leads to fractionation of DNA fragments in accordance with the tightness of DNA-protein bonds in situ. The fractions obtained were analysed by agarose gel electrophoresis and dot-hybridization technique with the use of various DNA probes.

[Identification of the stable DNA-matrix complex (type II) as a site of replication fork].

The two types of DNA-matrix complexes (the weak and tight ones, or type I and type II, respectively) identified in our previous work were studied with respect to their involvement in DNA replication. Nuclei isolated from human fibrosarcoma HT1080 cell line were treated with either restriction endonucleases or ultrasonic desintegrator and afterwards subjected to the triple-gradient Nucleoprotein--Celite chromatography. This permitted fractionation of nuclear DNA into fragments not attached, weakly attached, and tightly attached to the nuclear matrix (DNA 0, DNA I, and DNA II, respectively).

[Radioactive labeling of DNA with 3H-thymidine: effect of the internal irradiation on cell growth, chromatin structure and gene activity].

The growth of djungarian hamster fibroblasts 4/21 is inhibited by 3H-thymidine present in a culture medium in concentrations from 18.5 to 740 KBq/ml. As judged from the gradient elution of DNA from isolated nuclei (the nucleoprotein-celite chromatography), DNA fragmentation increases together with the increase in 3H-thymidine concentration and the decrease in the cell growth rate.

[A new method of DNA and RNA transfer onto nitrocellulose filters during blot hybridization].

A quick (1-2 hour) method of DNA and RNA transfer onto nitrocellulose filters for subsequent blot-hybridization was elaborated. The main features of the method proposed are, firstly, almost complete exclusion of the mechanical impact on the gel and, secondly, addition to the transfer medium (20 X SSC) of a chaotropic agent, 0.5 M NaClO4.

[Relation between the structural, metabolic and "adhesive" properties of nuclear and cytoplasmic non-ribosomal RNA].

Nuclear RNAs release from nucleoproteins of isolated nuclei absorbed on a celite column in a wide range of dissociating conditions (from 1 M LiCl--2 M urea at 2 degrees C to 4 M LiCl--8 M urea at 70-80 degrees C) was demonstrated. Such a high "adhesive" heterogeneity of nuclear RNAs (i.e.

[Hydroxyapatite-mediated transfer of DNA on nitrocellulose filters in dot-hybridization experiments].

The method of hydroxylapatite-mediated rapid and effective transfer of DNA onto nitrocellulose filters for following dot-hybridization was elaborated. The analysed DNA occurred initially in diluted and large volume solutions (from 1 to 10 ml) with various composition (2 M NaCl; 4 M LiCl--8 M urea; 4 M CsCl; 5 and 20% sucrose) was adsorbed on hydroxylapatite and quantitatively transferred onto nitrocellulose after hydroxylapatite solubilization in a small volume of acid (usually, 200 microliters of 10% TCA). As exemplified by the hybridization of total rat liver DNA with the plasmid ph22 DNA containing a cluster of sea urchin histone genes, the method presented appears to be not only simple and useful for handling multiple probes of diluted DNA solutions with high concentrations of salts, sucrose and urea but also more sensitive than some convenient DNA dot-hybridization methods.

[Analysis of cellular nucleoproteins by the nucleoprotein-celite chromatography method. III. Two types of DNA-nuclear matrix interaction].

There are two types of DNA-nuclear matrix interactions in animal cells as revealed by the release of DNA from isolated nuclei by three successive gradients: NaCl, LiCl-urea and temperature. Nuclei were treated with dissociating agents while being adsorbed on the Celite columns. "Weak" DNA-matrix interactions which dissociate in 1.

[Turnover of nuclear RNA. II. Conveyer model of synthesis and maturation of nuclear RNA as an alternative for a stochastic model].

It is shown that the stochastic model of hnRNA decay is inconsistent with a number of experimental data. The model of ordered in time and space movement of nascent and post-transcriptional RNA molecules "on conveyer" coupled with certain processing steps is put forward. Since degradation of the pre-mRNA molecule proceeds not instantaneously but in several subsequent steps, the notion of the "exiton" is introduced.

[Analysis of cellular nucleoproteins by nucleoprotein-celite-chromatography. I. Structural rearrangements of the chromosomal apparatus coupled with cellular quiescence-growth transitions].

By using the nucleoprotein-celite-chromatography of unfractionated cell lysates it was found that two alternative states of the chromosomal apparatus of eukaryotic cells exist, one of them being characteristic of resting cells (relaxed form), while another of growing cells (stabilized form). This finding evidences for the real occurrence of a special state of cellular quiescence, G0. DNA in form beta is much more tightly associated with proteins than DNA alpha.