Transient transfection
Transient transfection overview
Transient transfection of DNA into cells is an important technique to express foreign genes in mammalian cells for a short period of time (usually 24 to 72 hours). The foreign recombinant DNA will eventually be lost in daughter cells after mitosis since it isn't inserted into hosting cell genome.
General guidlines for transient transfection of foreign DNA into cells
1. Depending on cell types and gene of interests to be transfected, it is necessary to determine the harvest time interval after transient transfection.
2. Transiently transfected cells can be assayed at trascriptional (RNA) and translational (protein) level.
3. Transfection efficiency is often related to cell density at the time of transfection, cell type and passage numbers, DNA quality and should be optimized experimentally.
Transient transfection methods
1. DEAE-dextran cationic polymer transfection reagent.
2. Calcium phosphate (co-precipitation) transfection.
3. Cationic lipids transfection (most commonly used method).
4. Microinjection of DNA into cells.
5. Electroporation DNA transfection.
6. Viral transduction methods for high efficient gene delivery into primary cell culture (adenovirus, AAV, lentivirus etc).
Technical papers on gene transient transfection
1: Koyama S, Kimura T, Ogita K, Nakamura H, Tabata C, Md Abu Hadi Noor Ali K,
Temma-Asano K, Shimoya K, Tsutsui T, Koyama M, Kaneda Y, Murata Y.
Simple and highly efficient method for transient in vivo gene transfer to
mid-late pregnant mouse uterus.
J Reprod Immunol. 2006 Jun;70(1-2):59-69. Epub 2006 Feb 20.
2: Higashi T, Nagamori E, Sone T, Matsunaga S, Fukui K.
A novel transfection method for mammalian cells using calcium alginate
microbeads.
J Biosci Bioeng. 2004;97(3):191-5.
3: Mandell JW, Manabe R, Horwitz AF, Baumgart JP.
Fluorescence imaging of mobility shifts: an expression cloning method for
identification of cell signaling targets.
Lab Invest. 2002 Dec;82(12):1631-6.
4: Raykov Z, Legrand V, Homann HE, Rommelaere J.
Transient suppression of transgene expression by means of antisense
oligonucleotides: a method for the production of toxin-transducing recombinant
viruses.
Gene Ther. 2002 Mar;9(5):358-62.
5: Grant ER, Dubin AE, Zhang SP, Zivin RA, Zhong Z.
Simultaneous intracellular calcium and sodium flux imaging in human vanilloid
receptor 1 (VR1)-transfected human embryonic kidney cells: a method to resolve
ionic dependence of VR1-mediated cell death.
J Pharmacol Exp Ther. 2002 Jan;300(1):9-17.
6: Luo KQ, Yu VC, Pu Y, Chang DC.
Application of the fluorescence resonance energy transfer method for studying
the dynamics of caspase-3 activation during UV-induced apoptosis in living HeLa
cells.
Biochem Biophys Res Commun. 2001 May 25;283(5):1054-60.
7: Youil R, Toner TJ, Su Q, Kaslow DC.
Rapid method for the isolation of full length adenoviral genomes by bacterial
intermolecular homologous recombination.
J Virol Methods. 2001 Mar;92(1):91-7.
8: Newell JG, Davies M, Bateson AN.
The use of site-directed mutagenesis, transient transfection, and radioligand
binding. A method for the characterization of receptor-ligand interactions.
Mol Biotechnol. 2000 Jan;14(1):25-45.
9: Oral HB, Arancibia-Carcamo CV, Haskard DO, George AJ.
A method for determining the cytoprotective effect of catalase in transiently
transfected cell lines and in corneal tissue.
Anal Biochem. 1999 Feb 1;267(1):196-202.
10: Sawai K, Ohno K, Iijima Y, Levin B, Meruelo D.
A novel method of cell-specific mRNA transfection.
Mol Genet Metab. 1998 May;64(1):44-51.
11: Arbour NC, Ross TK, Zierold C, Prahl JM, DeLuca HF.
A highly sensitive method for large-scale measurements of 1,25-dihydroxyvitamin
D.
Anal Biochem. 1998 Jan 1;255(1):148-54.
12: Lauer U, Burgelt E, Squire Z, Messmer K, Hofschneider PH, Gregor M, Delius
M.
Shock wave permeabilization as a new gene transfer method.
Gene Ther. 1997 Jul;4(7):710-5.
13: Cheng L, Bucana CD, Wei Q.
Fluorescence in situ hybridization method for measuring transfection
efficiency.
Biotechniques. 1996 Sep;21(3):486-91.
14: Tomita N, Morishita R, Higaki J, Tomita S, Aoki M, Ogihara T, Kaneda Y.
In vivo gene transfer of insulin gene into neonatal rats by the HVJ-liposome
method resulted in sustained transgene expression.
Gene Ther. 1996 Jun;3(6):477-82.
15: Grimm T, Johnson JP.
A modified screening method for pcDNA-1 expression libraries which is
applicable to both surface and intracellular antigens. Cloning of a colon
carcinoma antigen.
J Immunol Methods. 1995 Oct 26;186(2):305-12.
16: McMahon SB, Norvell A, Levine KJ, Monroe JG.
Transient transfection of murine B lymphocyte blasts as a method for examining
gene regulation in primary B cells.
J Immunol Methods. 1995 Feb 27;179(2):251-9.
17: Yee JK, Miyanohara A, LaPorte P, Bouic K, Burns JC, Friedmann T.
A general method for the generation of high-titer, pantropic retroviral
vectors: highly efficient infection of primary hepatocytes.
Proc Natl Acad Sci U S A. 1994 Sep 27;91(20):9564-8.
18: Gabellini N, Manev RM, Manev H.
Is the heterologous expression of metabotropic glutamate receptors (mGluRs) an
appropriate method to study the mGluR function? Experience with human embryonic
kidney 293 cells transfected with mGluR1.
Neurochem Int. 1994 Jun;24(6):533-9.
19: Morales MJ, Gottlieb DI.
A polymerase chain reaction-based method for detection and quantification of
reporter gene expression in transient transfection assays.
Anal Biochem. 1993 Apr;210(1):188-94.
20: Parruti G, Lombardi MS, Chuang TT, De Blasi A.
Rhodopsin phosphorylation by transiently expressed human beta ARK1: a new
method for drug development.
J Recept Res. 1993;13(1-4):95-103.
21: Keyt BA, Berleau LT, Nguyen HV, Bennett WF.
Radioiodination of the active site of tissue plasminogen activator: a method
for radiolabeling serine proteases with tyrosylprolylarginyl chloromethyl
ketone.
Anal Biochem. 1992 Oct;206(1):73-83.
22: Kappes DJ, Strominger JL, Monos DS.
A novel method for generating stable high-level transfectants involving direct
immunomagnetic selection for cell-surface epitopes: expression of HLA class-II
genes in HeLa cells.
Gene. 1991 Dec 15;108(2):245-52.
23: Wakita K, Lord JM, Tokuhisa T.
A method for detecting the expression of a toxic gene in cultured cells.
Anal Biochem. 1991 Nov 1;198(2):224-7.
24: Levesque JP, Sansilvestri P, Hatzfeld A, Hatzfeld J.
DNA transfection in COS cells: a low-cost serum-free method compared to
lipofection.
Biotechniques. 1991 Sep;11(3):313-4, 316-8.
25: Oliveira MM, Barroso J, Pais MS.
Direct gene transfer into Actinidia deliciosa protoplasts: analysis of
transient expression of the CAT gene using TLC autoradiography and a GC-MS-based
method.
Plant Mol Biol. 1991 Aug;17(2):235-42.
26: Tatsuka M, Orita S, Yagi T, Kakunaga T.
An improved method of electroporation for introducing biologically active
foreign genes into cultured mammalian cells.
Exp Cell Res. 1988 Sep;178(1):154-62.
27: Presse F, Quillet A, Mir L, Marchiol-Fournigault C, Feunteun J, Fradelizi
D.
An improved electrotransfection method using square shaped electric impulsions.
Biochem Biophys Res Commun. 1988 Mar 30;151(3):982-90.
28: Gopal TV.
Gene transfer method for transient gene expression, stable transformation, and
cotransformation of suspension cell cultures.
Mol Cell Biol. 1985 May;5(5):1188-90.
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