Effect of fetal bovine serum on 3-methylcholanthrene-induced transformation of hamster cells in vitro

Summary Eighteen lots of fetal bovine serum were tested for their ability to support clonal growth and 3-methylcholanthrene-induced morphological transformation of hamster embryo cells in vitro. Most of them supported cloning efficiencies of over 11%. However, cloning efficiency alone was an inadequate criterion for selecting serum for transformation studies, since no transformation was observed with some lots, even though their cloning efficiencies were over 16%. This shows the importance of pretesting serum for its ability to support morphological transformation before it is used in mammalian cell carcinogenesis tests. Research sponsored by the National Cancer Institute under Contract No. N01-CO-75380 with Litton Bionetics, Inc.     

Effects of steroid hormones in fetal bovine serum on plating ang cloning of human cells in vitro.

Fetal bovine sera from each of three different commercial sources were tested for their ability to support cloning of human fibroblastoid cells in vitro. Cloning efficiencies varied according to serum source. Serum (10 samples) from company A did not support growth, while sera (10 samples) from companies B and C provided adequate to excellent conditions for cloning and growth. Cells from neonatal foreskin or embryonic lung responded to each serum similarly. Bovine serum albumin type H7 from company C supported cell growth in media without serum. Sera containing 1.0 ng per ml or more of progesterone inhibited growth, whereas sera containing less than 1.0 ng per ml supported cloning and growth. In the low progesterone sera, the concentration of 17-beta-estradiol exceeded 100 pg per ml. Growth supporting sera could be made non-supportive by adding 0.1 mug per ml of progesterone. The addition to non-supporative sera of 0.1 mug per ml of 17-beta-estradiol or hydrocortisone made these sera supportive of cell growth. Addition of estrogen or hydrocortisone to a culture medium that inhibits growth, with subsequent reversal of the inhibitory effect, implies that these hormones competitively regulate growth of responsive cells in vitro.     

Effects of steroid hormones in fetal bovine serum on plating and cloning of human cells in vitro

Summary Fetal bovine sera from each of three different commercial sources were tested for their ability to support cloning of human fibroblastoid cells in vitro. Cloning efficiencies varied according to serum source. Serum (10 samples) from company A did not support growth, while sera (10 samples) from companies B and C provided adequate to excellent conditions for cloning and growth. Cells from neonatal foreskin or embryonic lung responded to each serum similarly. Bovine serum albumin type H7 from company C supported cell growth in media without serum. Sera containing 1.0 ng per ml or more of progesterone inhibited growth, whereas sera containing less than 1.0 ng per ml supported cloning and growth. In the low progesterone sera, the concentration of 17-β-estradiol exceeded 100 pg per ml. Growth supporting sera could be made non-supportive by adding 0.1 μg per ml of progesterone. The addition to non-supportive sera of 0.1 μg per ml of 17-β-estradiol or hydrocortisone made these sera supportive of cell growth. Addition of estrogen or hydrocortisone to a culture medium that inhibits growth, with subsequent reversal of the inhibitory effect, implies that these hormones competitively regulate growth of responsive cells in vitro. Supported in part by NIH-NCI-EC2074.     

A simple and ultra-low cost homemade seamless ligation cloning extract (SLiCE) as an alternative to a commercially available seamless DNA cloning kit

The seamless ligation cloning extract (SLiCE) method is a novel seamless DNA cloning tool that utilizes homologous recombination activities in Escherichia coli cell lysates to assemble DNA fragments into a vector. Several laboratory E. coli strains can be used as a source for the SLiCE extract; therefore, the SLiCE-method is highly cost-effective.The SLiCE has sufficient cloning ability to support conventional DNA cloning, and can simultaneously incorporate two unpurified DNA fragments into vector. Recently, many seamless DNA cloning kits have become commercially available; these are generally very convenient, but expensive. In this study, we evaluated the cloning efficiencies between a simple and highly cost-effective SLiCE-method and a commercial kit under various molar ratios of insert DNA fragments to vector DNA. This assessment identified that the SLiCE from a laboratory E. coli strain yielded 30?85% of the colony formation rate of a commercially available seamless DNA cloning kit. The cloning efficiencies of both methods were highly effective, exhibiting over 80% success rate under all conditions examined. These results suggest that SLiCE from a laboratory E. coli strain can efficiently function as an effective alternative to commercially available seamless DNA cloning kits.     

Plating Efficiency for Primary Hamster Embryo Cells as an Index of Efficacy of Fetal Bovine Serum for Cell Culture

Attachment and growth of mammalian cells plated at low cell density require optimum conditions for the cells to form colonies. Reliability, reproducibility, and validity of the plating efficiency test for evaluating cell culture sera were determined by measuring the plating efficiency of 37 lots of fetal bovine serum obtained from 8 suppliers (5 lots from each of 7, 2 lots from 1 supplier), by using hamster embryo fibroblasts plated at low cell density. The test revealed considerable variation between lots of serum and between suppliers. The five lots from some suppliers had consistently high plating efficiencies, whereas one or more lots from other suppliers had quite low efficiencies. The results were reproducible in repeated tests, and control experiments indicated that the test measured the efficiency of the test serum independently of the efficiency of the serum used for the primary outgrowth of the hamster embryo cells.     

A comparative study on neoplastic transformation of human and Syrian hamster cells

Three approaches have been taken to study simultaneously Syrian hamster cells and human cells in order to develop an extrapolation from the more established hamster system to human cells. On the Characterization of normal cells in comparison to tumor cells, human tumor cells and hamster tumor cells showed similarity in displaying anchorage independence, growth in suspension as micro tumor spheroids, and xenotumorigenicity in contrast to their respective normal cells; in addition, these tumor cells exhibited shorter population doubling time, higher saturation density, higher cloning efficiency, and higher fibrinolytic activity relative to their respective normal cell types. Other differences including ploidy change, contact inhibition on growth, serum requirement, and morphological transformation were also noted between human and hamster cells. On the application of microcarrier culture for a xenotumorigenicity test, the microcarrier technique seemed to have enhanced the sensitivity by reducing the number of inoculated tumor cells required for tumor formation. On the in vitro transformation of normal human and hamster cells, the highest efficiency of morphological transformation of hamster cells has been observed in the group treated with N-methyl-N'-nitro-N-nitrosoguanidine followed by griseofulvin which was employed to enhance the transformation by disturbing the chromosome apparatus. However, no evidence of transformation was observed in the treated human cells thus far.     

Plasmid uptake by bacteria: a comparison of methods and efficiencies

The ability to introduce individual molecules of plasmid DNA into cells by transformation has been of central importance to the recent rapid advancement of plasmid biology and to the development of DNA cloning methods. Molecular genetic manipulation of bacteria requires the development of plasmid-mediated transformation systems that include (1) chemical transformation, (2) electro-transformation, (3) biolistic transformation, and (4) sonic transformation, leading to the introduction of exogenous plasmid DNA into bacterial cells. In this review, the manipulation properties and transformation efficiencies of these techniques are described. In addition to these methods, a conceptually novel transformation technique, namely the hydrogel exposure method, was developed. The hydrogel exposure method, based on the Yoshida effect, provides a significant advance over chemical means for transforming many strains of Escherichia coli and a variety of other bacterial species. The new term “tribos transformation” has been proposed for this novel technique. We also determined that, compared to conventional methods, the hydrogel exposure method is a novel and convenient method by which to transform bacteria.     

Transformation of homozygous diploid potato with an Agrobacterium tumefaciens binary vector system by adventitious shoot regeneration on leaf and stem segments

Transformed potato (Solanum tuberosum) plants were obtained from homozygous diploid potato by using a transformation procedure in combination with an adventitious shoot regeneration method. Leaf and stem explants were inoculated with an Agrobacterium tumefaciens strain which contained a binary vector (pVU 1011) carrying the neomycin phosphotransferase gene. Shoot regeneration most effectively on stem explants, occurred within six weeks directly from the explants without introducing a callus phase. A strong seasonal influence on transformation efficiencies was observed. Analysis of a number of randomly selected regenerated shoots for their ability to root and form shoots on kanamycin-containing medium shows that over 90% of the regenerated shoots obtained are transformed. In a number of shoots transformation was confirmed by a test for the presence and expression of the NPT-II gene.     

A procedure for the CHO/HGPRT mutation assay involving treatment of cells in suspension culture and selection of mutants in soft-agar

A procedure involving treatment of cells in suspension culture and soft-agar cloning was developed for measuring mutation of Chinese hamster ovary (CHO) cells to 6-thioguanine (6TG) resistance. The use of suspension cultures precluded the need for trypsinization and also permitted a 5-fold increase in cell population for compound exposure and mutant selection as compared to former monolayer techniques. Soft-agar cloning reduced the opportunity for metabolic cooperation and permitted the use of non-dialyzed fetal calf serum which resulted in spontaneous mutant frequencies of 6.6 +/- 3.2 X 10(-6) and cloning efficiencies of 91 +/- 18%. Relative total growth values were calculated based on suspension growth and cloning efficiencies such that an assessment of toxicity could be estimated from treatment through cloning. Dose-dependent mutagenic responses were observed in CHO cells following treatment with ethyl methanesulfonate, methyl methanesulfonate, 4-nitroquinoline-1-oxide, methylnitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine. Clones of 6TG-resistant cells harvested from soft agar maintained 6TG resistance and methotrexate sensitivity and did not incorporate [3H]hypoxanthine into DNA. These preliminary findings indicate that the use of suspension cultures and soft-agar cloning has improved the efficiency and cost effectiveness of the CHO/HGPRT mutation assay.     

Multifunctional poly(ethylene glycol) semi-interpenetrating polymer networks as highly selective adhesive substrates for bioadhesive peptide grafting

Novel artificial extracellular matrices were synthesized in the form of semi-interpenetrating polymer networks containing copolymers of poly(ethylene glycol) and acrylic acid (PEG-co-AA) grafted with synthetic bioadhesive peptides onto exposed carboxylic acid moieties. These substrates were very resistant to cell adhesion, but when they were grafted with adhesive peptides they were highly biospecific in their ability to support cell adhesion. Extensive preadsorption of adhesive proteins or peptides did not render these materials cell adhesive; yet covalent grafting of adhesive peptides did render these materials highly cell adhesive even in the absence of serum proteins. Polymer networks containing immobilized PEG-co-AA were grafted with peptides at densities of 475 +/- 40 pmol/cm(2). Polymer networks containing immobilized PEG-co-AA N-terminally grafted with GRGDS supported cell adhesion efficiencies of 42 +/- 4% 4 h after seeding and became confluent after 12 h. These cells displayed cell spreading and cytoskeletal grafted with inactive control peptides (GRDGS, GRGES, or no peptide) supported cell adhesion efficiencies of 0 +/- 0%, even when challenged with high seeding densities (to 100,000 cell/cm(2)) over 14 days. These polymer networks are suitable substrates to investigate in vitro cell-surface interactions in the presence of serum proteins without nonspecific protein adsorption adhesion signals other than those immobilized for study.     

Transformation of Aspergillus parasiticus with a homologous gene (pyrG) involved in pyrimidine biosynthesis

The lack of efficient transformation methods for aflatoxigenic Aspergillus parasiticus has been a major constraint for the study of aflatoxin biosynthesis at the genetic level. A transformation system with efficiencies of 30 to 50 stable transformants per microgram of DNA was developed for A. parasiticus by using the homologous pyrG gene. The pyrG gene from A. parasiticus was isolated by in situ plaque hybridization of a lambda genomic DNA library. Uridine auxotrophs of A. parasiticus ATCC 36537, a mutant blocked in aflatoxin biosynthesis, were isolated by selection on 5-fluoroorotic acid following nitrosoguanidine mutagenesis. Isolates with mutations in the pyrG gene resulting in elimination of orotidine monophosphate (OMP) decarboxylase activity were detected by assaying cell extracts for their ability to convert [14C]OMP to [14C]UMP. Transformation of A. parasiticus pyrG protoplasts with the homologous pyrG gene restored the fungal cells to prototrophy. Enzymatic analysis of cell extracts of transformant clones demonstrated that these extracts had the ability to convert [14C]OMP to [14C]UMP. Southern analysis of DNA purified from transformant clones indicated that both pUC19 vector sequences and pyrG sequences were integrated into the genome. The development of this pyrG transformation system should allow cloning of the aflatoxin-biosynthetic genes, which will be useful in studying the regulation of aflatoxin biosynthesis and may ultimately provide a means for controlling aflatoxin production in the field.     

In vitro development of bovine embryos as affected by different lots of bovine serum albumin and citrate

The effect of bovine serum albumin (BSA) lots on the development of in vitro-derived bovine embryos in synthetic oviductal fluid was investigated. Citrate concentration was determined for each lot of BSA, and then correlated with differences noted in the ability of BSA lots to support embryo development. Development of bovine embryos to the blastocyst stage was also compared after culture in chemically-defined medium with varying levels of citrate. There were distinct differences in the ability of the different BSA lots to support embryo development to the blastocyst stage (P相似文献   

Transformation of Aspergillus parasiticus with a homologous gene (pyrG) involved in pyrimidine biosynthesis.

The lack of efficient transformation methods for aflatoxigenic Aspergillus parasiticus has been a major constraint for the study of aflatoxin biosynthesis at the genetic level. A transformation system with efficiencies of 30 to 50 stable transformants per microgram of DNA was developed for A. parasiticus by using the homologous pyrG gene. The pyrG gene from A. parasiticus was isolated by in situ plaque hybridization of a lambda genomic DNA library. Uridine auxotrophs of A. parasiticus ATCC 36537, a mutant blocked in aflatoxin biosynthesis, were isolated by selection on 5-fluoroorotic acid following nitrosoguanidine mutagenesis. Isolates with mutations in the pyrG gene resulting in elimination of orotidine monophosphate (OMP) decarboxylase activity were detected by assaying cell extracts for their ability to convert [14C]OMP to [14C]UMP. Transformation of A. parasiticus pyrG protoplasts with the homologous pyrG gene restored the fungal cells to prototrophy. Enzymatic analysis of cell extracts of transformant clones demonstrated that these extracts had the ability to convert [14C]OMP to [14C]UMP. Southern analysis of DNA purified from transformant clones indicated that both pUC19 vector sequences and pyrG sequences were integrated into the genome. The development of this pyrG transformation system should allow cloning of the aflatoxin-biosynthetic genes, which will be useful in studying the regulation of aflatoxin biosynthesis and may ultimately provide a means for controlling aflatoxin production in the field.     

An interlaboratory comparison of enhanced morphological transformation of Syrian hamster embryo cells cultured under conditions of reduced bicarbonate concentration and pH

Initial studies performed in our laboratory indicated that early passage Syrian hamster embryo (SHE) cells exhibit optimal clonal proliferation when cultured in medium with a sodium bicarbonate concentration of 8.9 mM and pH of 6.70 instead of 44 mM and pH 7.35 as used previously by others. Subsequent studies indicated that morphological transformation frequency induced by benzo[a]pyrene (BP) was also enhanced at pH 6.70 compared to 7.35 and the level of enhancement was affected by cell density and duration of culture. With optimal conditions identified, the carcinogens BP, 3-methylcholanthrene, N-methyl-N'-nitro-N-nitrosoguanidine, 2-acetylaminofluorene and the non-carcinogen anthracene were tested at pH 6.70 and 7.35 in our laboratory and at Microbiological Assoc. Inc. under code. Additionally, the non-carcinogens 4-acetylaminofluorene, and caprolactam were tested in our laboratory. Results from these studies indicate that all carcinogens tested caused a significant increase in morphological transformation frequency compared to controls at pH 6.70. In contrast, only BP caused a significant increase in the morphological transformation frequency at pH 7.35. The non-carcinogens did not significantly increase the morphological transformation frequency compared to controls. Interlaboratory comparisons were in qualitative agreement despite the fact that different lots of serum and hamster cell isolates were used by the two laboratories. However, different dose-response curves for the various chemicals were observed between the two labs. It was also demonstrated that the enhanced morphological transformation frequency is not due to a decrease in culture medium osmolality or Na concentration, a condition which accompanies media with a reduced bicarbonate concentration and pH. These results demonstrate that the chemicals tested, low pH transformation of SHE cells agrees with carcinogenic potential and that assay variability is minimized. The implications of these results regarding use of the SHE cell assay as a short-term test for predicting the carcinogenic potential of chemicals are discussed.     

Cholesterol as a limiting factor in the growth of Mycoplasma pneumoniae.

Ultracentrifugation was used to separate three commercial lots of bovine serum fraction (BSF) into components designed to contain lipoproteins. Each BSF lot and component was tested for ability to support the growth of tree strains of Mycoplasma pneumoniae. In general, the level of growth-promoting activity corresponded to the amount of cholesterol present in the BSF or BSF components rather than to the amount or type of lipoprotein Cholesterol was the limiting nutritional factor of BSF with low growth-promoting activity. The addition of cholesterol and bovine serum albumin to BSF with low activity resulted in growth equal to or greater than that observed for BSF with high growth-promoting activity. When cholesterol was added to agar medium containing BSF of low activity, mycoplasma colonies were greater in number, possessed larger mean diameters, and had centers that were more distinct than those observed when this BSF was used alone. Variability in growth-promoting actions of commercial lots of BSF was eliminated by increasing their cholesterol content to an optimum level. An adjustment of the cholesterol and albumin levels of any serum product used in culture media may provide a simple convenient method to improve growth and isolation of mycoplasmas.     

Production of male cloned mice from fresh, cultured, and cryopreserved immature Sertoli cells

Although it is generally accepted that relatively high efficiencies of somatic cell cloning in mammals can be achieved by using donor cells from the female reproductive system (e.g., cumulus/granulosa, oviduct, and mammary gland cells), there is little information on the possibility of using male-specific somatic cells as donor cells. In this study we injected the nucleus of immature mouse Sertoli cells isolated from the testes of newborn (Days 3-10) males into enucleated mature oocytes in order to examine the ability of their nuclei to support embryonic development. After activation of the oocytes that had received the freshly recovered immature Sertoli cells, some developed into the morula/blastocyst stage, depending on the age of the donor cells (22.0-37.4%). When transferred into pseudopregnant females, 7 (3.3%, 7 of 215) developed into normal pups at term. Nuclear transfer of immature Sertoli cells after 1 wk in culture also produced normal pups after embryo transfer (3.1%, 2 of 65). Even after cryopreservation in a conventional cryoprotectant solution, their ability as donor cells was maintained, as demonstrated by the birth of cloned young (6.7%, 7 of 105). Immature Sertoli cells transfected with green fluorescent protein gene also supported embryo development into morulae/blastocysts, which showed specific fluorescence. This study demonstrates that immature Sertoli cells, male-specific somatic cells, are potential donors for somatic cell cloning.     

Genetic transformation of somatic cells. X. Morphological transformation and changes in the growth characteristics of NIH 3T3 cells administered the oncogene v-myc

Transfection of the plasmid containing the cloned gag-myc part of retrovirus MC 29 into mouse NIH 3T3 cells induces focuses of morphological transformation. Isolated morphological transformants have a decreased dependence on serum growth factors, a higher saturation density in monolayer, and an increased cloning efficiency on the glass and in agar. Induced traits are stably inherited, and may constitute the direct consequence of stable maintenance and expression of transfected oncogens.     

Neoplastic transformation of mast cells by Abelson-MuLV: abrogation of IL-3 dependence by a nonautocrine mechanism

Normal mast cells can be propagated in culture when medium is supplemented with interleukin-3 (IL-3). We demonstrate that Abelson-MuLV (Ab-MuLV) infection of mast cells eliminates dependence on IL-3 for growth. By contrast, Harvey, BALB, and Moloney MSV, which also productively infect mast cells, are unable to relieve IL-3 dependence. Ab-MuLV-induced IL-3-independent lines express the v-abl-specific transforming protein and have phenotypic characteristics of mast cells. These cells also possess high cloning efficiencies in soft agarose and are tumorigenic in nude mice. In addition, Ab-MuLV induces transplantable mastocytomas in pristane-primed adult mice resistant to lymphoid transformation, defining a new hematopoietic target for malignant transformation by this virus. None of the Ab-MuLV-derived transformants express or secrete detectable levels of IL-3 nor is their growth inhibited by anti-IL-3 serum. These results argue that Ab-MuLV abrogation of the IL-3 requirement is not due to an autocrine mechanism.     

Expression cloning of oncogenes by retroviral transfer of cDNA libraries.

a cDNA library transfer system based on retroviral vectors has been developed for expression cloning in mammalian cells. The use of retroviral vectors results in stable cDNA transfer efficiencies which are at least 100-fold higher than those achieved by transfection and therefore enables the transfer and functional screening of very large libraries. In our initial application of retroviral transfer of cDNA libraries, we have selected for cDNAs which induce oncogenic transformation of NIH 3T3 fibroblasts, as measured by loss of contact inhibition of proliferation. Nineteen different transforming cDNAs were isolated from a total of 300,000 transferred cDNA clones. Three of these cDNAs were derived from known oncogenes (raf-1, lck, and ect2), while nine others were derived from genes which had been cloned previously but were not known to have the ability to transform fibroblasts (beta-catenin, thrombin receptor, phospholipase C-gamma 2 and Spi-2 protease inhibitor genes). The Spi-2 cDNA was expressed in antisense orientation and therefore is likely to act as an inhibitor of an endogenous transformation suppressor. Seven novel cDNAs with transforming activities, including those for three new members of the CDC24 family of guanine nucleotide exchange factors, were also cloned from the retroviral cDNA libraries. Retroviral transfer of libraries should be generally useful for cloning cDNAs which confer selectable phenotypes on many different types of mammalian cells.     

Plasmid transformation of Streptococcus lactis protoplasts: optimization and use in molecular cloning.

The parameters affecting polyethylene glycol-induced plasmid transformation of Streptococcus lactis LM0230 protoplasts were examined to increase the transformation frequency. In contrast to spreading protoplasts over the surface of an agar medium, their incorporation into soft agar overlays enhanced regeneration of protoplasts and eliminated variability in transformation frequencies. Polyethylene glycol with a molecular weight of 3,350 at a final concentration of 22.5% yielded optimal transformation. A 20-min polyethylene glycol treatment of protoplasts in the presence of DNA was necessary for maximal transformation. The number of transformants recovered increased as the protoplast and DNA concentration increased over a range of 3.0 X 10(6) to 3.0 X 10(8) protoplasts and 0.25 to 4.0 micrograms of DNA per assay, respectively. With these parameters, transformation was increased to 5 X 10(3) to 4 X 10(4) transformants per microgram of DNA. Linear and recombinant plasmid DNA transformed, but at frequencies 10- to 100-fold lower than that of covalently closed circular DNA. Transformation of recombinant DNA molecules enabled the cloning of restriction endonuclease fragments coding for lactose metabolism into S. lactis LM0230 with the Streptococcus sanguis cloning vector, pGB301. These results demonstrated that the transformation frequency is sufficient to clone plasmid-coded genes which should prove useful for strain improvement of dairy starter cultures.