Nucleic Acids Research, Vol 24, Issue 4 596-601, Copyright © 1996 by Oxford University Press
M Jordan, A Schallhorn and FM Wurm
DNA-calcium phosphate co-precipitates arise spontaneously in supersaturated
solutions. Highly effective precipitates for transfection purposes,
however, can be generated only in a very narrow range of physico-chemical
conditions that control the initiation and growth of precipitate complexes.
The concentrations of calcium and phosphate are the main factors
influencing characteristics of the precipitate complex, but other
parameters, such as temperature, DNA concentration and reaction time are
important as well. An example for this is the finding that almost all of
the soluble DNA in the reaction mix can be bound into an insoluble complex
with calcium phosphate in <1 min. Extending the reaction time to 20 min
results in aggregation and/or growth of particles and reduces the level of
expression. With improved protocols we gained better reproducibility and
higher efficiencies both for transient and for stable transfections. Up to
60% of cells stained positive for beta-gal and transient production of
secreted proteins was improved 5- to 10-fold over results seen with
transfections using standard procedures. Similar improvements in efficiency
(number of recombinant cell colonies) were observed with stable
transfections, using co-transfected marker plasmids for selection.
Transient expression levels 2 days after DNA transfer and titers obtained
from stable cell lines, emerging weeks later, showed strong correlation.
ARTICLES
Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation
Genentech Inc., South San Francisco, CA 94080, USA.
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K. A. Kovacs, M. Steinmann, P. J. Magistretti, O. Halfon, and J.-R. Cardinaux CCAAT/Enhancer-binding Protein Family Members Recruit the Coactivator CREB-binding Protein and Trigger Its Phosphorylation J. Biol. Chem., September 19, 2003; 278(38): 36959 - 36965. [Abstract] [Full Text] [PDF]
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L. Chen, L. Smith, Z. Wang, and J. B. Smith Preservation of Caspase-3 Subunits from Degradation Contributes to Apoptosis Evoked by Lactacystin: Any Single Lysine or Lysine Pair of the Small Subunit Is Sufficient for Ubiquitination Mol. Pharmacol., August 1, 2003; 64(2): 334 - 345. [Abstract] [Full Text] [PDF]
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M. Ogrunc and A. Sancar Identification and Characterization of Human MUS81-MMS4 Structure-specific Endonuclease J. Biol. Chem., June 6, 2003; 278(24): 21715 - 21720. [Abstract] [Full Text] [PDF]
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C. R. Brandt, A. W. Kolb, D. D. Shah, A. M. Pumfery, R. L. Kintner, E. Jaehnig, and J. J. Van Gompel Multiple Determinants Contribute to the Virulence of HSV Ocular and CNS Infection and Identification of Serine 34 of the US1 Gene as an Ocular Disease Determinant Invest. Ophthalmol. Vis. Sci., June 1, 2003; 44(6): 2657 - 2668. [Abstract] [Full Text] [PDF]
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S. Chantalat, R. Courbeyrette, F. Senic-Matuglia, C. L. Jackson, B. Goud, and A. Peyroche A Novel Golgi Membrane Protein Is a Partner of the ARF Exchange Factors Gea1p and Gea2p Mol. Biol. Cell, June 1, 2003; 14(6): 2357 - 2371. [Abstract] [Full Text] [PDF]
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T. Nakamura, M. Sano, Z. Songyang, and M. D. Schneider A Wnt- and beta -catenin-dependent pathway for mammalian cardiac myogenesis PNAS, May 13, 2003; 100(10): 5834 - 5839. [Abstract] [Full Text] [PDF]
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N. Petit, A. Lescure, M. Rederstorff, A. Krol, B. Moghadaszadeh, U. M. Wewer, and P. Guicheney Selenoprotein N: an endoplasmic reticulum glycoprotein with an early developmental expression pattern Hum. Mol. Genet., May 1, 2003; 12(9): 1045 - 1053. [Abstract] [Full Text] [PDF]
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G. K. Seabold, A. Burette, I. A. Lim, R. J. Weinberg, and J. W. Hell Interaction of the Tyrosine Kinase Pyk2 with the N-Methyl-D-aspartate Receptor Complex via the Src Homology 3 Domains of PSD-95 and SAP102 J. Biol. Chem., April 18, 2003; 278(17): 15040 - 15048. [Abstract] [Full Text] [PDF]
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D. Dulude, M. Baril, and L. Brakier-Gingras Characterization of the frameshift stimulatory signal controlling a programmed -1 ribosomal frameshift in the human immunodeficiency virus type 1 Nucleic Acids Res., December 1, 2002; 30(23): 5094 - 5102. [Abstract] [Full Text] [PDF]
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F. Legros, A. Lombes, P. Frachon, and M. Rojo Mitochondrial Fusion in Human Cells Is Efficient, Requires the Inner Membrane Potential, and Is Mediated by Mitofusins Mol. Biol. Cell, December 1, 2002; 13(12): 4343 - 4354. [Abstract] [Full Text] [PDF]
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L. Smith and J. B. Smith Lack of Constitutive Activity of the Free Kinase Domain of Protein Kinase C zeta . DEPENDENCE ON TRANSPHOSPHORYLATION OF THE ACTIVATION LOOP J. Biol. Chem., November 22, 2002; 277(48): 45866 - 45873. [Abstract] [Full Text] [PDF]
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 W. El-Sankary, V. Bombail, G. G. Gibson, and N. Plant Glucocorticoid-Mediated Induction of CYP3A4 is Decreased by Disruption of a Protein: DNA Interaction Distinct from the Pregnane X Receptor Response Element Drug Metab. Dispos., September 1, 2002; 30(9): 1029 - 1034. [Abstract] [Full Text] [PDF]
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D. A. Bennin, A. S. A. Don, T. Brake, J. L. McKenzie, H. Rosenbaum, L. Ortiz, A. A. DePaoli-Roach, and M. C. Horne Cyclin G2 Associates with Protein Phosphatase 2A Catalytic and Regulatory B' Subunits in Active Complexes and Induces Nuclear Aberrations and a G1/S Phase Cell Cycle Arrest J. Biol. Chem., July 19, 2002; 277(30): 27449 - 27467. [Abstract] [Full Text] [PDF]
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A.-L. Ducrest, M. Amacker, J. Lingner, and M. Nabholz Detection of promoter activity by flow cytometric analysis of GFP reporter expression Nucleic Acids Res., July 15, 2002; 30(14): e65 - e65. [Abstract] [Full Text] [PDF]
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 D. L. Cioffi, T. M. Moore, J. Schaack, J. R. Creighton, D. M.F. Cooper, and T. Stevens Dominant regulation of interendothelial cell gap formation by calcium-inhibited type 6 adenylyl cyclase J. Cell Biol., June 24, 2002; 157(7): 1267 - 1278. [Abstract] [Full Text] [PDF]
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Y. Yang, B. Dong, P. R. Mittelstadt, H. Xiao, and J. D. Ashwell HIV Tat Binds Egr Proteins and Enhances Egr-dependent Transactivation of the Fas Ligand Promoter J. Biol. Chem., May 24, 2002; 277(22): 19482 - 19487. [Abstract] [Full Text] [PDF]
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M. Ibberson, B. M. Riederer, M. Uldry, B. Guhl, J. Roth, and B. Thorens Immunolocalization of GLUTX1 in the Testis and to Specific Brain Areas and Vasopressin- Containing Neurons Endocrinology, January 1, 2002; 143(1): 276 - 284. [Abstract] [Full Text] [PDF]
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W. El-Sankary, G. G. Gibson, A. Ayrton, and N. Plant Use of a Reporter Gene Assay to Predict and Rank the Potency and Efficacy of CYP3A4 Inducers Drug Metab. Dispos., November 1, 2001; 29(11): 1499 - 1504. [Abstract] [Full Text] [PDF]
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 A.-L. Ducrest, M. Amacker, Y. D. Mathieu, A. P. Cuthbert, D. A. Trott, R. F. Newbold, M. Nabholz, and J. Lingner Regulation of Human Telomerase Activity: Repression by Normal Chromosome 3 Abolishes Nuclear Telomerase Reverse Transcriptase Transcripts but Does Not Affect c-Myc Activity Cancer Res., October 1, 2001; 61(20): 7594 - 7602. [Abstract] [Full Text] [PDF]
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L. A. Lindsey-Boltz, V. P. Bermudez, J. Hurwitz, and A. Sancar Purification and characterization of human DNA damage checkpoint Rad complexes PNAS, September 25, 2001; 98(20): 11236 - 11241. [Abstract] [Full Text] [PDF]
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 S. Cestele, T. Scheuer, M. Mantegazza, H. Rochat, and W. A. Catterall Neutralization of Gating Charges in Domain II of the Sodium Channel {alpha} Subunit Enhances Voltage-Sensor Trapping by a {beta}-Scorpion Toxin J. Gen. Physiol., September 1, 2001; 118(3): 291 - 302. [Abstract] [Full Text] [PDF]
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T. C. Rich, T. E. Tse, J. G. Rohan, J. Schaack, and J. W. Karpen In Vivo Assessment of Local Phosphodiesterase Activity Using Tailored Cyclic Nucleotide-Gated Channels as Camp Sensors J. Gen. Physiol., July 1, 2001; 118(1): 63 - 78. [Abstract] [Full Text] [PDF]
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R. Yu, V. Hebbar, D. W. Kim, S. Mandlekar, J. M. Pezzuto, and A.-N. T. Kong Resveratrol Inhibits Phorbol Ester and UV-Induced Activator Protein 1 Activation by Interfering with Mitogen-Activated Protein Kinase Pathways Mol. Pharmacol., July 1, 2001; 60(1): 217 - 224. [Abstract] [Full Text]
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K. Jeyaseelan, D. Ma, and A. Armugam Real-time detection of gene promoter activity: quantitation of toxin gene transcription Nucleic Acids Res., June 15, 2001; 29(12): e58 - e58. [Abstract] [Full Text] [PDF]
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S. A. Mitchell, E. C. Brown, M. J. Coldwell, R. J. Jackson, and A. E. Willis Protein Factor Requirements of the Apaf-1 Internal Ribosome Entry Segment: Roles of Polypyrimidine Tract Binding Protein and upstream of N-ras Mol. Cell. Biol., May 15, 2001; 21(10): 3364 - 3374. [Abstract] [Full Text]
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D. J. Orlicky and J. Schaack Adenovirus transduction of 3T3-L1 cells J. Lipid Res., March 1, 2001; 42(3): 460 - 466. [Abstract] [Full Text]
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 R. M. Roman, R. L. Smith, A. P. Feranchak, G. H. Clayton, R. B. Doctor, and J. G. Fitz ClC-2 chloride channels contribute to HTC cell volume homeostasis Am J Physiol Gastrointest Liver Physiol, March 1, 2001; 280(3): G344 - G353. [Abstract] [Full Text] [PDF]
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C. Herrera, V. Casadó, F. Ciruela, P. Schofield, J. Mallol, C. Lluis, and R. Franco Adenosine A2B Receptors Behave as an Alternative Anchoring Protein for Cell Surface Adenosine Deaminase in Lymphocytes and Cultured Cells Mol. Pharmacol., January 1, 2001; 59(1): 127 - 134. [Abstract] [Full Text]
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W. El-Sankary, N. J. Plant, G. G. Gibson, and D. J. Moore Regulation of the CYP3A4 Gene by Hydrocortisone and Xenobiotics: Role of the Glucocorticoid and Pregnane X Receptors Drug Metab. Dispos., May 1, 2000; 28(5): 493 - 496. [Abstract] [Full Text]
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A. Bollig and R. J. Miksicek An Estrogen Receptor-{alpha} Splicing Variant Mediates Both Positive and Negative Effects on Gene Transcription Mol. Endocrinol., May 1, 2000; 14(5): 634 - 649. [Abstract] [Full Text]
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R. Yu, S. Mandlekar, S. Ruben, J. Ni, and A-N. T. Kong Tumor Necrosis Factor-related Apoptosis-inducing Ligand-mediated Apoptosis in Androgen-independent Prostate Cancer Cells Cancer Res., May 1, 2000; 60(9): 2384 - 2389. [Abstract] [Full Text]
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