Efficient recruitment of transfected DNA to a homologous chromosomal target in mammalian cells.

A Chinese hamster ovary cell line hemizygous for a defective adenine phosphoribosyltransferase (aprt) gene was transfected with a plasmid, pAG100, capable of correcting the endogenous aprt mutation by targeted homologous recombination. In some experiments, pAG100 was transfected in combination with one of two 'competitor' plasmids. Competitor pCOMP-A was identical to pAG100 except that the aprt sequence on pCOMP-A had the same mutation as the endogenous aprt gene. Competitor pCOMP-B was identical to pAG100 except for a 763 bp deletion in the aprt sequence encompassing the site of mutation in the endogenous gene. Neither pCOMP-A nor pCOMP-B was capable of correcting the defect in the endogenous aprt gene via gene targeting. We asked whether cotransfection of a 4-fold excess of either competitor DNA molecule with pAG100 would reduce the efficiency of targeted correction of the endogenous aprt gene. We report that while plasmid pCOMP-B did not influence the efficiency of gene targeting by pAG100, plasmid pCOMP-A reduced the number of gene targeting events about 5-fold. These observations indicate that the initial homologous interaction between transfected DNA and a genomic target sequence occurs rapidly and that targeting efficiency is limited by a step subsequent to homologous pairing.

Stable transfection of mammalian cells by syringe-mediated mechanical loading of DNA.

We show that mammalian cells can be stably transfected by a mechanical loading procedure in which cells are forced through a small opening in the presence of DNA. A suspension of cells and plasmid DNA in growth medium was passed up and down through a 30-gauge needle attached to a 1-ml syringe. Cells were immediately plated at appropriate densities for subsequent selection for stable expression of a marker gene. Two rodent cell lines, Chinese hamster ovary and mouse Ltk- cells, were successfully transfected with an efficiency of about one transfectant per 5 x 10(4) cells. The human HeLa cell line was transfected with a somewhat lower efficiency. Pluronic F-68, a detergent believed to aid in healing of membrane injuries, had no beneficial effect when present during the loading procedure. Successful transfection was accomplished using three different genes as selectable markers. Southern blotting analysis revealed that transfectants contained one or very few copies of the introduced DNA construct integrated into the genome. Several transfectants were demonstrated to remain stable for more than 20 generations of growth in the absence of selection. This procedure is fast, economical, and of general utility.

Enrichment for gene targeting in mammalian cells by inhibition of poly(ADP-ribosylation).

Inhibition of poly(ADP-ribosylation) reduces random genomic integration of transfected DNA and mildly stimulates intrachromosomal homologous recombination in mammalian cells. We investigated the effect of inhibition of poly(ADP-ribosylation) on the efficiency of gene targeting in Chinese hamster ovary (CHO) cell line ATS-49tg. This cell line is hemizygous for a defective adenine phosphoribosyltransferase (aprt) gene and is hypoxanthine phosphoribosyltransferase (hprt) deficient. Plasmid pAG100 contains a portion of the CHO aprt gene sufficient to correct the defect in ATS-49tg cells via gene targeting; pAG100 also contains an Escherichia coli guanine phosphoribosyltransferase (gpt) gene. Following transfection of ATS-49tg cells with pAG100, selection for gpt-positive transfectants allowed recovery of cells that had randomly integrated pAG100 while selection for aprt-positive cells allowed recovery of cells that had undergone gene targeting at the endogenous aprt locus. Treatment of cells with 3 mM 3-methoxybenzamide (3-MB), an inhibitor of poly(ADP-ribose) polymerase, decreased random integration and gene targeting of electroporated pAG100 about 5-fold. In contrast, treatment with 3 mM 3-MB during calcium phosphate transfection could reduce random integration more than 150-fold while reducing gene targeting less than two-fold. Therefore, as much as a 100-fold enrichment for gene targeting was achieved with calcium phosphate transfection.

Stimulation of intrachromosomal homologous recombination in mammalian cells by an inhibitor of poly(ADP-ribosylation).

We determined the effect of 3-methoxybenzamide (3-MB), a competitive inhibitor of poly(ADP-ribose) polymerase (E.C. 2.4.2.30), on intrachromosomal homologous recombination in mouse Ltk- cells. We used a cell line that contained in its genome two defective Herpes thymidine kinase (tk) genes as closely linked direct repeats. Intrachromosomal homologous recombination events were monitored by selecting for tk-positive segregants that arose during propagation of the cells and recombination rates were determined by fluctuation analysis. We found that growth of cells in the continuous presence of 2mM 3-MB increased intrachromosomal recombination between 3 and 4-fold. Growth of cells in the presence of 2mM m-anisic acid, a non-inhibitory analog of 3-MB, had no effect on intrachromosomal recombination rates. Additionally, we found that 3-MB increased both gene conversions and crossovers to similar extents, adding to the evidence that these two types of intrachromosomal rearrangements share a common pathway. These findings contrast with our previous studies [Waldman, B.C. and Waldman, A.S. (1990) Nucleic Acids Res., 18, 5981-5988] in which we determined that 3-MB inhibits illegitimate recombination and has no effect on extrachromosomal homologous recombination in mouse Ltk- cells. An hypothesis is offered that explains the influence of 3-MB on different recombination pathways in mammalian cells in terms of the role that poly(ADP-ribosylation) plays in DNA break-repair.

Illegitimate and homologous recombination in mammalian cells: differential sensitivity to an inhibitor of poly(ADP-ribosylation).

We determined the effect of 3-methoxybenzamide (3-MB), a competitive inhibitor of poly(ADP-ribose)polymerase (E.C. 2.4.2.30), on illegitimate and extrachromosomal homologous recombination in mouse Ltk- cells. Cells were transfected with a wild type Herpes thymidine kinase (tk) gene or with two defective tk gene sequences followed by selection for tk-positive colonies. Using a wild type tk gene, colony formation required uptake, integration, and expression of the tk gene. Using defective tk genes, colony formation had the additional requirement for homologous recombination to reconstruct a functional tk gene. The presence of non-cytotoxic levels of 3-MB during and after transfection reduced the number of colonies recovered with a wild type tk gene in a dose-dependent manner, with 2 mM 3-MB causing a 10 to 20-fold reduction. 3-MB reduced the number of colonies recovered with defective tk genes only to the same extent as in transfections with a wild type gene. Treatment with 3-methoxybenzoic acid, a non-inhibitory analog of 3-MB, did not reduce the recovery of colonies in any experiment. Similar results were obtained using linear or supercoiled molecules and when defective tk genes were transfected into cells on one or two different DNA molecules. By assaying for transient expression of the tk gene, we found that 3-MB did not inhibit uptake or expression of the tk gene. We conclude that poly(ADP-ribosylation) plays a role in random integration (illegitimate recombination) of DNA but does not play an important role in extrachromosomal homologous recombination, demonstrating that these two recombination pathways in cultured mouse fibroblasts are biochemically distinct.

UDP-GlcNAc transport across the Golgi membrane: electroneutral exchange for dianionic UMP.

We have examined the coupling and charge stoichiometry for UDP-GlcNAc transport into Golgi-enriched vesicles from rat liver. In the absence of added energy sources, these Golgi vesicles concentrate UDP-GlcNAc at least 20-fold, presumably by exchange with endogenous nucleotides. Under the conditions used, extravesicular degradation of UDP-GlcNAc has been eliminated, and less than 15% of the internalized radioactivity becomes associated with endogenous macromolecules. Of the remaining intravesicular label, 85% remains unmetabolized UDP-[3H]GlcNAc, and approximately 15% is hydrolyzed to [3H]GlcNAc-1-phosphate. Efflux of accumulated UDP-[3H]GlcNAc is induced by addition of UMP, UDP, or UDP-galactose to the external medium. Permeabilization of Golgi vesicles causes a rapid and nearly complete loss of internal UDP-[3H]GlcNAc, indicating that the results reflect transport and not binding. Moreover, transport of UDP-[3H]GlcNAc into these Golgi vesicles was stimulated up to 5-fold by mechanically preloading vesicles with either UDP-GlcNAc or UMP. The response of UMP/UMP exchange and UMP/UDP-GlcNAc exchange to alterations in intravesicular and extravesicular pH suggests that UDP-GlcNAc enters the Golgi apparatus in electroneutral exchange with the dianionic form of UMP.

A method for replacing intravesicular contents of Golgi vesicles using an air-driven ultracentrifuge.

Golgi membrane vesicles can be easily and very rapidly (within 10 min.) loaded with solutions of desired composition by centrifugation of the vesicles at high g force in an air-driven ultracentrifuge and subsequent resuspension of the vesicle pellet. This centrifugal/mechanical loading procedure does not destroy the integrity of these vesicles, as demonstrated by the ability of loaded vesicles to (i) retain their contents, (ii) maintain a K+ gradient when loaded with K+ ions, and (iii) exchange internal UMP for external [3H]UMP when loaded with UMP. When radiolabeled solutes are loaded into vesicles, the displaced internal volume can be measured using a rapid filtration assay. This simple and rapid technique of replacing the intravesicular contents of Golgi membrane vesicles should prove useful in studying transport across this membrane and may have a variety of other applications, such as intravesicular volume measurements, macromolecule and drug delivery protocols, and the study of membrane fusion events.

A clonal derivative of tunicamycin-resistant Chinese hamster ovary cells with increased N-acetylglucosamine-phosphate transferase activity has altered asparagine-linked glycosylation.

A population of Chinese hamster ovary (CHO) cells resistant to the antibiotic tunicamycin (TM) had previously been isolated (Criscuolo, B.A., and Krag, S.S. (1982) J. Cell Biol. 94:586-591) by a stepwise selection procedure using progressive increments of TM added to the medium. TM inhibits asparagine-linked glycoprotein biosynthesis by blocking the transfer of N-acetylglucosamine-1-phosphate from the sugar nucleotide UDP-N-acetylglucosamine to the isoprenoid lipid carrier, dolichyl phosphate. Four clonal derivatives were isolated from the TM-resistant population in the presence of 27 micrograms TM/ml and were found to overproduce the N-acetylglucosamine-phosphate transferase activity to the same extent (approximately 15-fold compared to wild-type cells). One of these clones, 3E11, was greater than 550-fold more resistant to TM than wild-type cells. The resistance phenotype remained during at least 2.5 months of growth in the absence of TM. 3E11 cells exhibited chromosomal translocations, but no homogeneously staining regions (HSR) or double minute chromosomes. The N-acetylglucosamine-phosphate transferase activity in 3E11 cells was membrane-associated and was inhibited by TM. A 140,000-dalton membrane protein and at least four other membrane proteins were enriched in 3E11 cells. Mannosylphosphoryldolichol synthase and glucosylphosphoryldolichol synthase activities were not elevated in membranes prepared from 3E11 cells. Asparagine-linked glycosylation was altered such that 3E11 cells synthesized primarily a truncated oligosaccharide, Man5GlcNAc2, perhaps due to the reduced amount of mannosylphosphoryldolichol relative to wild-type cells.