We describe a simple calcium phosphate transfection protocol and neo marker vectors that achieve highly efficient transformation of mammalian cells. In this protocol, the calcium phosphate-DNA complex is formed gradually in the medium during incubation with cells and precipitates on the cells. The crucial factors for obtaining efficient transformation are the pH (6.95) of the buffer used for the calcium phosphate precipitation, the CO2 level (3%) during the incubation of the DNA with the cells, and the amount (20 to 30 micrograms) and the form (circular) of DNA. In sharp contrast to the results with circular DNA, linear DNA is almost inactive. Under these conditions, 50% of mouse L(A9) cells can be stably transformed with pcDneo, a simian virus 40-based neo (neomycin resistance) marker vector. The NIH3T3, C127, CV1, BHK, CHO, and HeLa cell lines were transformed at efficiencies of 10 to 50% with this vector and the neo marker-incorporated pcD vectors that were used for the construction and transduction of cDNA expression libraries as well as for the expression of cloned cDNA in mammalian cells.

High-efficiency transformation of mammalian cells by plasmid DNA.

Drug sensitivity assays were performed using a variation of a colorimetric [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)] assay on V79, CHO-AuxB1, CHRC5, NCI-H460, and NCI-H249 cell lines following optimization of experimental conditions for each cell line. Results from this assay were compared with data assimilated simultaneously by clonogenic assay and by dye exclusion assay. Good correlation was observed using the CHO-AuxB1 cell line and the pleiotropic drug-resistant mutant CHRC5, with similar degrees of relative resistance observed with both the MTT and clonogenic assays. Good correlation was observed between the clonogenic and MTT assays for 1-h drug exposures, although the MTT assay was more sensitive to vinblastine. In general, the clonogenic assay was more sensitive when continuous drug exposures were utilized, although this was primarily related to the increased drug exposure time. While the use of the MTT assay in drug sensitivity testing of primary tumor samples is limited, since contaminating normal cells may also reduce the tetrazolium, the MTT assay can be semiautomated, and therefore it offers a valid, simple method of assessing chemosensitivity in established cell lines.

https://cancerres.aacrjournals.org/content/canres/47/4/936.full.pdf

Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing.

It is concluded from a review of the literature that plant cell culture itself generates genetic variability (somaclonal variation). Extensive examples are discussed of such variation in culture subclones and in regenerated plants (somaclones). A number of possible mechanisms for the origin of this phenomenon are considered. It is argued that this variation already is proving to be of significance for plant improvement. In particular the phenomenon may be employed to enhance the exchange required in sexual hybrids for the introgression of desirable alien genes into a crop species. It may also be used to generate variants of a commercial cultivar in high frequency without hybridizing to other genotypes.

Somaclonal variation — a novel source of variability from cell cultures for plant improvement

Mouse bone marrow hematopoietic stem cells were isolated with the use of a variety of phenotypic markers. These cells can proliferate and differentiate with approximately unit efficiency into myelomonocytic cells, B cells, or T cells. Thirty of these cells are sufficient to save 50 percent of lethally irradiated mice, and to reconstitute all blood cell types in the survivors.

https://science.sciencemag.org/content/sci/241/4861/58.full.pdf

Purification and Characterization of Mouse Hematopoietic Stem Cells

Internal duplication and homology with bacterial transport proteins in the mdr1 (P-glycoprotein) gene from multidrug-resistant human cells

The Effect of Differing Demands for Blood Cell Production on DNA Synthesis by Hemopoietic Colony-Forming Cells of Mice

On the nature of hereditable variation in cultured somatic cells.

Ouabain-resistant mutants of mouse and hamster cells in culture

A two-stage procedure has been used to obtain hemopoietic spleen colonies derived from single precursor cells containing radiation-induced chromosomal markers. Of a total of 46 colonies examined, 17 were found to contain cells with abnormal karyotypes. In each of the 17 marked colonies, 90% or more of the dividing cells in the colony carried the same marker. Cell suspensions prepared from each of the individual colonies were tested for their content of dividing cells possessing recognizable differentiated functions. Metaphase cells with peroxidase-positive granules in their cytoplasm were considered to be members of the granulopoietic series, while metaphase cells which contained Fe55 were considered to be members of the erythropoietic series. Results were obtained for 12 of the marked colonies, and in nine of these, the percentage of metaphases lacking the marker was less than the percentage of metaphases which were scored as erythropoietic, and also was less than the percentage of metaphases scored as granulopoietic. This is the result which would be expected if the marker were present in both erythropoietic and granulopoietic cells. These results provide support for the view that colony forming hemopoietic stem cells are multipotent, and that differentiation along more than one pathway can occur during the formation of macroscopic splenic colonies.

Louis Siminovitch

Papers

The Effect of Differing Demands for Blood Cell Production on DNA Synthesis by Hemopoietic Colony-Forming Cells of Mice

On the nature of hereditable variation in cultured somatic cells.

Ouabain-resistant mutants of mouse and hamster cells in culture

A cytological study of the capacity for differentiation of normal hemopoietic colony-forming cells.

Selection and characterization of eight phenotypically distinct lines of lectin-resistant chinese hamster ovary cells