Hiroto Okayama

High-efficiency cloning of full-length cDNA.

A widely recognized difficulty of presently used methods for cDNA cloning is obtaining cDNA segments that contain the entire nucleotide sequence of the corresponding mRNA. The cloning procedure described here mitigates this shortcoming. Of the 10(5) plasmid-cDNA recombinants obtained per microgram of rabbit reticulocyte mRNA, about 10% contained a complete alpha- of beta-globin mRNA sequence, and at least 30 to 50%, but very likely more, contained the entire globin coding regions. We attribute the high efficiency of cloning full- or nearly full-length cDNA to (i) the fact that the plasmid DNA vector itself serves as the primer for first- and second-strand cDNA synthesis, (ii) the lack of any nuclease treatment of the products, and (iii) the fact that one of the steps in the procedure results in preferential cloning of recombinants with full-length cDNAs over those with truncated cDNAs.

Primary structure of rat chromogranin A and distribution of its mRNA.

The primary structure of rat chromogranin A has been deduced from a rat adrenal cDNA clone. A comparison of rat and bovine chromogranin A reveals several similar features: clusters of polyglutamic acid, similar amino acid composition, position of seven of 10 pairs of basic amino acids, identical placement of the only two cysteine residues, a highly conserved N‐ and C‐terminus, and a sequence homologous to porcine pancreastatin 1–49 [(1986) Nature 324, 476–478]. Unique features of rat chromogranin A are an eicosaglutamine sequence and two potential N‐linked glycosylation sites. Chromogranin A mRNA is detectable in adrenal medulla, anterior pituitary, cerebral cortex, and hippocampus, as well as tumor cell lines derived from pancreas, pituitary, and adrenal medulla.

Bacteriophage lambda vector for transducing a cDNA clone library into mammalian cells.

We have developed a bacteriophage lambda vector (lambda NMT) that permits efficient transduction of mammalian cells with a cDNA clone library constructed with the pcD expression vector (H. Okayama and P. Berg, Mol. Cell. Biol. 3:280-289, 1983). The phage vector contains a bacterial gene (neo) fused to the simian virus 40 early-region promoter and RNA processing signals, providing a dominant-acting selectable marker for mammalian transformation. The phage DNA can accommodate pcD-cDNA recombinants with cDNA of up to about 9 kilobases without impairing the ability of the phage DNA to be packaged in vitro and propagated in vivo. Transfecting cells with the lambda NMT-pcD-cDNA recombinant phage yielded G418-resistant clones at high frequency (approximately 10(-2]. Cells that also acquired a particular cDNA segment could be detected among the G418-resistant transformants by a second selection or by a variety of screening protocols. Reconstitution experiments indicated that the vector could transduce 1 in 10(6) cells for a particular phenotype if the corresponding cDNA was present as 1 functional cDNA clone per 10(5) clones in the cDNA library. This expectation was confirmed by obtaining two hypoxanthine-guanine phosphoribosyltransferase (HPRT)-positive transductants after transfecting 10(7) HPRT-deficient mouse L cells with a simian virus 40-transformed human fibroblast cDNA library incorporated into the lambda NMT phage vector. These transductants contained the human HPRT cDNA sequences and expressed active human HPRT.

Calcium Phosphate Mediated Gene Transfer into Established Cell Lines

DNA transfection is one of the most important techniques in molecular genetics. It is this technique that has made possible the dissection of complex eukaryotic genes and the characterization of the function of their components (1-7) as well as the isolation of particular genes on the basis of their expression in cultured cells (8-11).

Suppression of the chemically transformed phenotype of BHK cells by a human cDNA.

Transformation of the baby hamster kidney cell line BHK SN-10 by chemical carcinogens such as nitrosylmethylurea (NMU) is mediated by the loss of a gene product critical for the suppression of malignant transformation. Somatic cell hybrids between chemically transformed BHK SN-10 cells and either normal hamster kidney or human fibroblast cells are nontransformed; therefore, a recessive mechanism underlies the malignant transformation of BHK SN-10 cells after chemical carcinogenesis (A. Stoler and N. P. Bouck, Proc. Natl. Acad. Sci. USA 82:570-574, 1985). A human fibroblast cDNA library was constructed and introduced into NMU-transformed BHK SN-10 cells (NMU 34m) in order to identify a human cDNA capable of suppressing cellular transformation. NMU-transformed BHK cells were analyzed for reversion to an anchorage-dependent normal cellular phenotype after transfection with human cDNA. The human cDNA capable of inducing stable reversion of NMU 34m cells encodes the intermediate filament protein vimentin, which is apparently required for maintenance of the normal phenotype in BHK SN-10 cells.

Calcium phosphate transfection.

This unit contains two methods of calcium phosphate‐based eukaryotic cell transfection, protocols that can be used for both transient and stable transfections. In the protocols, plasmid DNA is introduced to monolayer cell cultures via a precipitate that adheres to the cell surface. The uses a HEPES‐buffered solution to form a calcium phosphate precipitate that is directly layered onto the cells. In the alternate high‐efficiency method, a BES‐buffered system is used that allows the precipitate to form gradually in the medium and is then dropped onto the cells. The alternate method is particularly efficient for stable transformation of cells with circular plasmid DNA, and may be helpful with linear or genomic DNA. Both methods of transfection require very high‐quality plasmid DNA, which can be prepared as described in the second . Transfection efficiency in some cell lines can be increased by shocking the cells with glycerol or dimethyl sulfoxide (DMSO) as described in the first .

Calcium Phosphate Transfection

This unit presents two methods of calcium phosphate‐based eukaryotic cell transfection that can be used for both transient and stable transfections. In these protocols, plasmid DNA is introduced to monolayer cell cultures via a precipitate that adheres to the cell surface. A HEPES‐buffered solution is used to form a calcium phosphate precipitate that is directly layered onto the cells. For some cells, shocking the cells with glycerol or DMSO improves transfection efficiency. In the alternate high‐efficiency method, a BES‐buffered system is used that allows the precipitate to form gradually in the medium and then drop onto the cells. While the alternate method is particularly efficient for stable transformation of cells with circular plasmid DNA, both protocols yield similar results for transformation with linear plasmid or genomic DNA, or for transient expression.