高效的酵母完整细胞转化法

以穿梭质粒PYES2转化酿酒酵母H158,用二硫苏糖醇（DTT）和处理酵母细胞,变性单链鱼精DNA（ssDNA）为质粒DNA的携带体;转化率比普通酵母完整细胞转化法提高200倍,可达8．3×104个转化子/μg质粒DNA。     

A novel method for transformation of intact yeast cells by electroinjection of plasmid DNA

Summary It was found that plasmid DNA (YEp13) can be introduced into intact yeast cells by electric field pulses. The frequency of transformation by this electroinjection method depend upon the initial electric field intensity, the capacitance of the electric discharge capacitor, and the number of pulses applied. A maximum number of transformants (90±20/gDNA) was obtained by three successive pulses with an initial intensity of 5 KV/cm and with a capacitance of 1 F. The present electroinjection method is simple, and transformants can be obtained within 2 to 3 days after transformation treatment.     

Factors enhancing genetic transformation of intact yeast cells modify cell wall porosity

Genetic transformation of intact cells of Saccharomyces cerevisiae, achieved by incubating the cells with plasmid DNA in the presence of PEG, could be enhanced, not only by pretreatment of the cells with Li+ and 2-mercaptoethanol, as has been reported previously, but also by pretreatment with proteolytic enzymes. The efficiency of transformation with 2-mercaptoethanol rose dramatically when the pretreated cells had been handled in osmotically stabilized media. Following all the pretreatments the cells became leaky for nucleic acids as detected by the presence of endogenous RNAs in the medium. The pretreatments evidently facilitated the passage of transforming DNA across the cell wall.     

Improved electroporation efficiency of intact Lactococcus lactis subsp. lactis cells grown in defined media.

The impact of growth conditions on electroporation of Lactococcus lactis subsp. lactis LM0230 (previously designated Streptococcus lactis LM0230) was evaluated. Cells grown in M17 broth supplemented with 0.5% glucose (M17-Glu) and two chemically defined synthetic media, FMC and RPMI 1640, all supplemented with 0.24% DL-threonine or 0.5% glycine, were harvested, washed with double-distilled water, diluted, and porated in the presence of 1 microgram of pGB301 DNA with a Transfector 100 (BTX, Inc., San Diego, Calif.) or a Gene Pulser (Bio-Rad Laboratories, Richmond, Calif.). Transformants were recovered at consistently higher efficiencies for cells grown in FMC or RPMI 1640 (10(3) to 10(4) transformants per micrograms of DNA) than for cells grown in M17-Glu (10(1) to 10(2) transformants per micrograms of DNA). Other parameters influencing electroporation of L. lactis cells grown in chemically defined media were growth phase and final concentration of cells, concentration of plasmid DNA, voltage achieved during poration, and expression conditions. A high degree of variability in transformation efficiencies was evident for replicate samples of cells pulsed with either electroporation machine. A trend toward decreased variability was observed for duplicate samples of cells prepared on the same day. In addition, storage studies done with a large batch of cells prepared on the same day indicated that freezing dry cell pellets at -60 degrees C had no deleterious effect on transformation efficiencies over a 30-day period when a new 0.2-cm cuvette was used for porating each sample.     

Improved electroporation efficiency of intact Lactococcus lactis subsp. lactis cells grown in defined media

The impact of growth conditions on electroporation of Lactococcus lactis subsp. lactis LM0230 (previously designated Streptococcus lactis LM0230) was evaluated. Cells grown in M17 broth supplemented with 0.5% glucose (M17-Glu) and two chemically defined synthetic media, FMC and RPMI 1640, all supplemented with 0.24% DL-threonine or 0.5% glycine, were harvested, washed with double-distilled water, diluted, and porated in the presence of 1 microgram of pGB301 DNA with a Transfector 100 (BTX, Inc., San Diego, Calif.) or a Gene Pulser (Bio-Rad Laboratories, Richmond, Calif.). Transformants were recovered at consistently higher efficiencies for cells grown in FMC or RPMI 1640 (10(3) to 10(4) transformants per micrograms of DNA) than for cells grown in M17-Glu (10(1) to 10(2) transformants per micrograms of DNA). Other parameters influencing electroporation of L. lactis cells grown in chemically defined media were growth phase and final concentration of cells, concentration of plasmid DNA, voltage achieved during poration, and expression conditions. A high degree of variability in transformation efficiencies was evident for replicate samples of cells pulsed with either electroporation machine. A trend toward decreased variability was observed for duplicate samples of cells prepared on the same day. In addition, storage studies done with a large batch of cells prepared on the same day indicated that freezing dry cell pellets at -60 degrees C had no deleterious effect on transformation efficiencies over a 30-day period when a new 0.2-cm cuvette was used for porating each sample.     

Electroporation-induced transformation of intact cells of Clostridium perfringens.

Electroporation-induced transformation of intact cells of Clostridium perfringens 3624A with plasmids pAMB1 and pHR106 resulted in 3.8 X 10(-5) and 4.2 X 10(-4) transformants per viable cell, respectively. With respect to shuttle plasmid pHR106, these values represent a greater than 100-fold increase in transformation frequency when compared with the values reported with polyethylene glycol-induced L-phase variants.     

A method for high efficiency YAC lipofection into murine embryonic stem cells.

We describe a modified protocol for introducing yeast artificial chromosomes (YACs) into murine embryonic stem (ES) cells by lipofection. With a decreased DNA:cell ratio, increased concentration of condensing agents and altered culture conditions, this protocol reduces the requirement for YAC DNA to a few micrograms, improves the recovery of neomycin-resistant ES colonies and increases the yield of clones containing both flanking vector markers and insert. These modifications enable generation of sufficient 'intact' transgenic clones for biological analysis with a single experiment.     

Genetic transformation of somatic cells. I. Clone of Chinese hamster cells defective in thymidine kinase and characterized by high transformation efficiency

A characteristics is given of clone A238 of the Chinese hamster cells deficient in thymidine kinase (TK). The isolation procedure is described. Upon transformation with the aid of DNA of plasmids, containing thymidine kinase gene (tk-gene) of Herpes simplex virus type 1 (HSV1) clone A238 cells show frequency (7.10(-5) and efficiency (130 TK+ colonies per 1 microgram of plasmid DNA) compatible with those of mouse line LMtk- cells. Modified transformation and selection conditions of clone A238 cells expressing TK-gene of HSV1 are demonstrated. A simple method is described for discriminating somatic cells, expressing either their proper or a virus TK-gene according to the cloning efficiency of cells on the HAT medium containing thymidine in concentration 100 micrograms/ml. It is shown that at the fixed total DNA concentrations a complete replacement of the eukaryotic carrier DNA for the plasmid DNA, containing no tg gene of HSV1, decreases but only insignificantly the frequency and efficiency of transformation.