experimentalist to retain authentic biological regulation of a recombinant cistron postintegration. What is the molecular basis of the enhancer effect? We know that enhancers dramatically affect transcription even when they are placed at great distances from their cognate promoters. We also know that, in many cases, enhancer multimers function more efficiently than the same enhancer when present only as a monomer, m Enhancer binding proteins have been isolated and we know, through the conduct of cis/trans tests, that these sequence-specific enhancer binding proteins are required for efficient enhancer function. We can assume that a multimeric enhancer binds more enhancer binding proteins than does the corresponding monomer. The simplest explanation for the action at a distance enhancer effect is that the enhancer binding protein-DNA complex directly interacts with the transcription complex, including RNA polymerase and other promoter binding proteins, at the promoter. The simplest mechanism by which this could be accomplished is to bend the DNA into a loop structure in a manner such that the enhancer binding complex is brought into direct physical contact with the appropriate promoter binding complex. Such an interaction might allow for the formation of a more stable, more easily regulatable multimolecular enhancer-promoter binding complex. As for the molecular mechanism of promoter-intron cross talk, that remains a mystery.

[41] M e t h o d s

for Introducing DNA into Mammalian Cells


S. K U C H E R L A P A T I

The ability to introduce defined genetic information into mammalian cells by various methods has revolutionized various aspects of the study of gene structure-function relationships. These methods can be classified as direct or indirect methods. Direct methods involve the introduction of genetic material (usually DNA) into the nucleus of a somatic cell, or the male pronucleus of a fertilized egg, by microinjection. Indirect methods involve the active or passive uptake of the genetic information by the cell which is to be transfected. The indirect methods most commonly used include delivery of genetic information by viral vectors, formation of complexes between DNA and chemical agents followed by active cellular uptake, or physical methods of introduction of nucleic acids into cells. The use of viral vectors, particularly that of viral RNA vectors, has become METHODS IN ENZYMOLOGY, VOL, 185

Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.



[41 ]

quite popular and has been dealt with extensively in recent articles. ~-a The discussion of procedures in this article is restricted to direct methods and to physical or chemical-mediated indirect methods for gene transfer. Chemical-mediated procedures discussed herein include formation of complexes of DNA with calcium phosphate, DEAE-dextran, Polybrene, or natural or synthetic cationic lipids. Physical methods include electric field-mediated DNA transfer (electroporation) and a microprojectile method in which DNA packaged into a "projectile" is "fired" into cells. Detailed protocols are described for some of the more popular procedures. The relative advantages of other procedures are also discussed. Specific protocols for these less widely used methodologies may be obtained from the references cited for each procedure. Calcium P h o s p h a t e Coprecipitation One of the most commonly used methods of introducing DNA into mammalian cells is to coprecipitate the DNA with calcium phosphate and present the mixture to cells. The technique was originally used to increase the infectivity of adenoviral DNA, a and was made popular by Wigler and colleagues4 and Maitland and McDougall. 5 Since the methods were originally described a number of permutations on the basic theme have been described. All of these procedures involve the mixing of purified DNA with buffers containing phosphate and calcium chloride which results in the formation of a very fine precipitate, and the presentation of this mixture to cells in culture. A typical protocol for cells which grow attached to a substratum follows: 1. On day 1, seed 2 - 3 × 104 cells/cm2 in normal growth medium. Allow the cells to attach. At the time of transfection the cells should be 80- 90% confluent. 2. On day 2, prepare the DNA-calcium phosphate coprecipitate: DNA x/zl TE 440 -- x/zl 2)< HBS 500/A 2 M CaCI2 60 #1 TE: 10 mMTris, 1 m M E D T A pH 8.0 i H. Temin, in "Gene Transfer" (R. Kucherlapati, ed.), p. 149. Plenum, New York, 1986. 2 V. R. Baichwal and B. Sugden, in "Gene Transfer" (R. Kucherlapati, ed.), p. 117. Plenum, New York, 1986. 3 F. L. Graham and A. J. Van Der Eb, Virology 52, 456 (1973). 4 M. Wiglet, S. Silverstein, L. S. Lee, A. Pellicer, V. C. Cheng, and R. Axel, Cell ll, 223 (1977). 5 N. J. Maitland and J. K. McDougall, Cell 11, 233 (1977).




2X HBS: Hanks' balanced salts, 1.4 m M Na2HPO4, 10 m M KC1, 12 m M glucose, 275 m M NaCI, and 40 m M HEPES, pH 6.95 2 M CaC12: calcium chloride in 10 m M HEPES, pH 5.8 Mix and allow to stand at room temperature for 30 min. 3. Remove medium from cells and replace with fresh medium (60-mm dish: 3.5 ml, 100-mm dish: 9 ml). 4. Mix the precipitate gently by shaking or pipetting and add directly to the medium in dishes containing cells (0.5 ml/60-mm dish, 1 ml/100-mm dish). 5. Incubate the cells at 37 ° for 4 hr. 6. Remove the medium containing the precipitate. Add 2 ml of 1020% (w/v) dimethyl sulfoxide (DMSO) in I X HBS. After 2 rain, add 4 ml of serum-free medium to each dish. Aspirate the mixture, wash the cell layer twice with serum-free medium, and add medium (4 ml/60-mm dish, 10 ml/100-mm dish). Incubate overnight at 37*. 7. Trypsinize the cells and split the contents of each plate into 3 - 4 fresh plates. If selection is applied for stable transfectants, selective medium can be used at this time or a day later. Notes. (1) The quality of the cells is important. Use rapidly dividing cultures for experiments. (2) The pH of the 2X HBS used to prepare the precipitate is critical. (3) The quality of the precipitate that is optimal for transfection can be assessed visually. It should be a hazy white suspension. If DNA precipitates to the bottom of the tube, repurify the DNA by protease digestion and phenol extraction before preparing another precipitate. (4) The time of incubation of the cells with the precipitate can be varied. Four hours is usually the minimum time. (5) Treatment of cells with DMSO or glycerol facilitates uptake of the DNA. The concentrations of these chemicals (usually 10-20%) and the time of treatment that are optimal vary from cell line to cell line. Use a concentration that is sublethal to cells. The time for which the cells are exposed to the chemical is crucial. If they are allowed to stay too long, cell viability may be dramatically reduced. If the cells are overly sensitive to these chemicals, they can be left with the DNA precipitate for 8 - 2 4 hr followed by a serum-free medium wash, omitting the chemical shock.

Using a variety of different cell types, we have obtained transfection efficiencies of up to 10-a. A modified calcium phosphate transfection protocol which yielded as much as 10- 15% or more stable tranfectants has been described by Chen and Okayama. 6 They indicate that the following factors played a role in the increased transfection efficiencies: 6 C. Chen and H. Okayama, Mol. Cell. Biol. 7, 2745 0987).




1. Temperature and CO2 levels: The precipitate is allowed to form as the cells are incubated at 35 ° in 2 - 4 % CO2 for 15-24 hr. 2. DNA concentration: A much finer precipitate is observed when the concentration of DNA is at 2 0 - 30 gg/ml than at other DNA concentrations. 3. DNA form: Circular DNA is far more active in this protocol than is linear DNA. 4. Nature of vector: A promoter-enhancer system which permits high levels of expression of the gene is found to be an efficient vector system. No chemical shock is employed in this protocol.




DEAE-dcxtran, which was initiallyused to increase viral infectivityof cells, was modified for transfcction of D N A into mammalian cells.This method is ideally suited for experiments in which transfcction is used for transient biological assays. The procedure is simple and efficient,but has not been shown to result in stable transfcction.7,s As with calcium phosphate coprccipitation procedures, m a n y variations on the procedure have evolved. The basic protocol used in our laboratory is a variation of the procedures of Lopata et al? and Sussman and Milman. i° It is described below. 1. On day 1, seed cells to be tranfected into tissue culture plates at a cell density of 13,000/cm 2, or at a density such that the monolayer will be about 90% confluent by the next day. 2. On day 2, to a sterile tube add DNA (usually 5 - 50 #g), DEAE-dextran (MW = 500,000) from a sterile 100 mg/ml stock, and serum-free medium and mix. The final concentration of DEAE-dextran should be 200 gg/ml and the final concentration of DNA should be 0.5- 6.5 gg/ml. 3. Remove medium from cells and wash once with serum-free medium. Add D N A - DEAE-dextran mixture directly onto the cell monolayer (50 gl for each em 2 of monolayer surface). Incubate at 37 ° for 4 hr. 4. Remove DNA-DEAE-dextran mixture from dishes and wash once with serum-free medium. Shock with 10% DMSO in 1 × HBS for 2 min. Wash with an equal volume of serum-free media three times. Add normal growth medium and incubate at 37 ° . 5. Assay 48-72 hr after transfection. vj. H. MeCutehanand J. S. Pagano,J. Natl. Cancer Inst. 41, 351 (1968). 8G. Milmanand M. Herzberg,Somatic Cell Genet. 7, 161 (1981). 9 M. A. Lopata,D. W. Cleveland,and B. Sollner-Webb,Nucleic Acids Res. 12, 5707 (1984). 1oD. J. Sussmanand G. Milman,Mol. Cell. Biol. 4, 1641 (1984).




Notes. ( 1) Because of the rather small volume of D N A - DEAE-dextran used, the plates should be incubated in a humidified atmosphere. (2) As with calcium phosphate transfections, the sensitivity of cells to DMSO or glycerol must be determined empirically. (3) This method has been reported to yield transfection frequencies as high as 80%. (4) This method is not useful to isolate stable transfectants. (5) DNA introduced into cells by this method and by calcium phosphate coprecipitation is subject to rather high rates of mutation. H- t3

Electric Field-Mediated DNA Transfection (Electroporation) Electric field-mediated DNA transfection, commonly called electroporation, is rapidly becoming the method of choice for many transfection applications. Advantages presented by this procedure include ease of operation, reproducibility of conditions, applicability to cells which grow attached or in suspension, utility for both stable and transient transfection procedures, and the capacity to control the copy number of transfected DNA molecules. When membranes are subjected to an electric field of sufficiently high voltage, regions of the membrane undergo a reversible breakdown, resulting in the formation of pores large enough to permit the passage of macromolecules. Unlike calcium phosphate coprecipitation, in which DNA entering the cell is taken up into phagocytic vesicles, t4 electroporated DNA remains free in the cytosol and nucleoplasm. ~5 Perhaps because of this difference, lower mutation frequencies are often observed for electroporated DNA than for DNA introduced into cells by calcium phosphate coprecipitation, t6 Many cell types which are resistant to transfection by other procedures are readily transfected by electroporation, including lymphocytes, ~7hematopoietic stem cells, t8 and rat hepatoma cells. ~9 By altering the parameters 1~ M. P. Calos, J. S. Lebkowski, and M. R. Botchan, Proc. Natl. Acad. Sci. U.S.A. 80, 3015 (1983). 12 A. Razzaque, H. Mizusawa, and M. M. Seidman, Proc. Natl. Acad. Sci. U.S.A. 80, 3010 (1983). 13 C. R. Ashman and R. L. Davidson, Somatic CellMol. Genet. 11,499 (1985). 14F. L. Graham and A. Van der Eb, Virology52, 456 (1973). t5 W. Bertling, K. Hunger-Bertling, and M. J. Cline, J. Biochem. Biophys. Methods 14, 223 (1987). t6 N. R. Drinkwater and D. K. Klinedinst, Proc. Natl. Acad. Sci. U.S.A. 83, 3402 (1986). t7 H. Potter, L. Weir, and P. Leder, Proc. Natl. Acad. Sci. U.S.A. 81, 7161 (1984). ts F. Toneguzzo and A. Keating, Proc. Natl. Acad. Sci. U.S.A. 83, 3496 (1986). 19C. Sureau, J.-L. Romet-Lemonne, J. I. Mullins, and M. Essex, Ce1147, 37 (1986).




for specific experiments, it is also possible to introduce as little as one or a few copies of the tranfected DNA by electroporation. 2° Exponentially growing cells are harvested and pelleted, then washed once with electroporation buffer (140 m M NaC1, 20 m M HEPES at pH 7.15, 750 # M Na2HPO4) and resuspended in electroporation buffer at a cell concentration of 2 - 2 0 X 106 cells/ml. DNA is added to the cell suspension, which is then incubated on ice for 10 min. The cell and DNA suspension is then subjected to an electric field and returned to ice for 10 min prior to plating in nonselective medium. Selection (or transient assay) can be carried out 48 hr later. The strength of the electric field appropriate for each cell type must be determined empirically. For most cell types we have tested, optimal transfection occurs at a field strength which results in a cell death of 50% or more with the conditions described. This generally corresponds to a capacitance of about 1 mF, a pulse time of 100 msec, and a potential of 100-400 V. Many units are commercially available which allow the experimeter to adjust the different parameters as desired. Notes. Several investigators have examined the effects of various parameters on levels of stable transfection obtained by this p r o c e d u r e . 2°-22 Results from these experiments indicate that a variety of conditions yield successful electroporation. Refer to these articles for specific conditions. P o l y b r e n e - M e d i a t e d DNA Transfection Polybrene-mediated DNA transfection, first described as a method for efficiently transfecting chick embryo fibroblasts with cloned Rous sarcoma virus DNA, 23 has more recently been optimized for stable transfection of CHO cells with DNA from a variety of sources. 24 The major advantages of this procedure over calcium phosphate coprecipitation are a 15-fold better stable transfection frequency in CHO cells and elimination of a requirement for carrier DNA to maximize transfection efficiency. Although the procedure also yields stable transfectants in other cell types, such as HeLa and L cells, it has not been shown to improve transfection frequencies in these cell types over those obtained with calcium phosphate methods. It is not yet clear whether the procedure can be optimized in cell lines other than CHO to yield improved transfection frequencies. 20 S. S. Boggs, R. G. Gregg, N. Borenstein, and O. Smithies, Exp. Hematol. 14, 988 (1986). 21 G. Chu, a . Hayakawa, and P. Berg, Nucleic Acids Res. 15, 131 i (1987). 22 R. Tur-Kaspa, L. Teicher, B. J. Levine, A. I. Skoultchi, and D. A. Shafritz, Mol. Cell. Biol 6, 716 (1986). 23 S. Kawai and M. Nishizawa, Mol. Cell. Biol. 4, 1172 (1984). 24 W. G. Chancy, D. R. Howard, J. W. Pollard, S. Sallustio, and P. Stanley, Somatic Cell Mol. Genet. 12, 237 (1986).




In the protocol described by Chaney et al., 24 5 X 105 CHO cells are plated in alpha medium containing 10% (v/v) fetal calf serum (FCS) and 3 gg/ml Polybrene in 100-mm tissue culture dishes and incubated at 37 ° overnight. The medium is removed and replaced with 3 ml of alpha medium containing DNA and 10 #g/ml Polybrene. Plates are gently rocked every 1.5 hr. After 12 hr, the Polybrene-DNA mixture is removed and the cells are shocked with DMSO (30% in FCS) for 4 rain, washed once with alpha medium, and fed with fresh alpha medium containing 10% FCS. Selection is initiated 24-48 hr later. Using this procedure with the shuttle vector pSV2neo, Chancy observed a transfection frequency of 36,000 G418-resistant colonies/pg plasmid DNA for 5 × 105 cells plated. The system also worked well for transfection of an amplified cosmid library DNA and total genomic DNA. High transfection efficiencies were obtained with l 0 - 30 ng DNA/dish, and the addition of carder DNA did not result in an increase in transfection efficiency. Lipid-Mediated Transfection A number of techniques based on the use oflipids have been developed. The best established of these methods utilizes unilamellar phospholipid vesicles (liposomes) which have been loaded with DNA. 25-27 This approach relies on the fusion of the DNA-containing vesicles with the plasma membrane of the recipient cells. The DNA appears to traverse the cytoplasm and subsequently enter the nucleus.2s This technique has been shown to be effective in both transient and stable expression, and has been used with both adherent and suspension cell types. A major drawback to this approach is the complexity of liposome preparation techniques. While the transfection efficiency of the liposome-mediated methods is not generally greater than that obtained by the other methods, such as calcium phosphate coprecipitation, it may nevertheless be useful for certain cell types which are difficult to transfect with other techniques. The ability to produce liposomes of roughly physiological composition reduces toxic effects observed with other protocols. A particularly exciting aspect of this method is the ability to perform in vivo transfections. Nicolau and colleagues29 have demonstrated transient expression of a preproinsulin gene 25 R. Fraley, S. Subramani, P. Berg, and D. Papahadjopoulous, J. Biol. Chem. 255, 10431 (1980). 26 T. K. Wong, C. Nicolau, and P. H. Hofschneider, Gene 10, 87 (1980). 27 M. Schaefer-Ridder, Y. Wang, and P. H. Hofschneider, Science 215, 166 (1982). 2s T. Itani, H. Arigg, N. Yamaguchi, T. Tadakuma, and T. Yasuda, Gene 56, 267 (1987). 29 C. Nicolau, A Lepapo, P. Soriano, F. Fargette, and M. F. Juhel, Proc. Natl. Acad. Sci. U.S.A. 80, 1068 (1983).




in the liver and spleen of rats injected with DNA-loaded liposomes. Three different methods which have been successfully used for the production of DNA-loaded unilamellar vesicles (liposomes) are (1) the ether-infusion method, 3° (2) reversed-phase evaporation method,al and (3) phosphatidylserine calcium-induced fusion method. 32

Lipofection A novel technique which also relies on the use of DNA-containing liposomes to introduce genetic material into cultured cells has been described. This protocol, referred to as lipofection, utilizes a synthetic cationic lipid, N-J1-(2, 3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). This molecule spontaneously associates with DNA and, on sonication, forms unilameUar vesicles in which 100% of the DNA is trapped. 33 Feigner et al. 33 described high levels of transient expression of introduced DNA in three different cell lines. Transfection efficiencies were 6 - 1 0 times greater than those obtained with DEAE-dextran, and stable transfection efficiencies which are 6 - 8 0 times greater than those obtained with calcium phosphate precipitation have been reported. The major advantage of this technique over the other liposome-mediated transfection techniques is the apparently greater transfection efficiency as well as the quantitative incorporation of DNA into liposomes (the previously described techniques incorporated only approximately 10% of DNA into the liposomes). However, the potential toxicity of the DOTMA may reduce the general usefulness of this method.

R e d Blood Cell-Mediated Transfection The extremely plastic structure of the erythrocyte and the ability to remove its cytoplasmic contents and reseal the plasma membrane have led a number of investigators to trap different macromolecules within the so-called hemoglobin free "ghost." Combining these "ghosts" with target cells and a fusogen such as polyethylene glycol has permitted the introduc3o D. Deamer and A. D. Bangham, Biochim. Biophys. Acta 443, 629 (1976). 3~ F. Szoka, Jr., and D. Papahadjopoulos, Proc. Natl. Acad. Sci. U.S.A. 75, 4194 (1978). 32 D. Papahadjopoulos, W. J. Vail, K. Jacobson, and G. Poste, Biochim. Biophys. Acta 394, 483 (1975). 33 D. L. Feigner, T. R. Gadek, M. Holm, R. Roman, H. W. Chan, M. Wenz, J. P. Northrop, G. M. Ringold, and M. Danielsen, Proc. Natl. Acad. Sci. U.S.A. 84, 7413 (1987).




tion o f a variety o f macromolecules into m a m m a l i a n cells. 34-~ Both transient and stable expression o f introduced D N A have been achieved by this method. 37-39 The complexities of obtaining and preparing erythrocyte ghosts and the variability of D N A entrapment obtained with this procedure have precluded its widespread use. DNA Microinjection As the name implies, this technique involves the direct microinjection o f DNA into the nucleus o f recipient cells. Unlike the other methods described in this article, microinjection does not expose the D N A to the cytoplasm or organelles within it. This is considered beneficial since it has been suggested that D N A sustains considerable damage during the transit from the cell exterior to the nucleus? ° Diacumakos and colleagues4~ first described microinjection o f a variety o f substances into h u m a n cultured cells. Calxcchi 42 described the use o f this approach to transform thymidine kinase-deficient ( T K - ) mouse cells with the herpes simplex virus T K gene. A particularly useful application o f D N A microinjection is in the production of transgenic mice. 43 Microinjection into mouse embryos has largely been replaced by the m e t h o d developed by G o r d o n et al., "~ in which the DNA is injected into the male pronucleus o f the fertilized mouse egg. D N A sequences introduced in this m a n n e r are detectable and expressable in some o f the progeny animals. Although the method requires use o f sophisticated equipment and technical expertise, it is very useful in cases where high efficiencies o f 34M. Furasawa, T. Nishimura, M. Yamaizumi, and Y. Okada, Nature (London) 249, 449 (1974). 35K. Kaitofi, J. Zeuthen, F. Engbaek, P. W. Piper, and J. E. Celis, Proc. Natl. Acad. Sci. U.S.A. 73, 2793 (1976). 36C. Boogardand G. H. Dixon, Exp. CellRes. 143, 175 (1983). 37F. C. Wiberg, P. Sunnerhagen, K. Kaltoft, J. Zeuthen, and G. Bjursell,Nucleic Acids Res. 11, 7287 (1983). 3s F. C. Wiberg, P. Sunnerhagen, and G. Bjursell,Mol. Cell. Biol. 6, 653 (1986). 39F. C. Wiberg, P. Sunnerhagen, and G. Bjursell,Exp. CellRes. 173, 218 (1987). 4oC. T. Wake, T. Gudewicz, T. Porter, A. White, and J. A. Wilson, Mol. Cell. Biol. 4, 387 (1984). 41E. G. Diacumakos, S. Holland, and P. Pecora, Proc. Natl. Acad. Sci. U.S.A. 65, 911 (1970). 42M. R. Capecchi, Cell 22, 479 (1980). 43M. L. De Pamphilis, S. A. Herman, E. Martinez-Salas,C. E. Chalifour, D. O. Wirak, D. Y. Cupo, and M. Miranda, BioTechniques 6, 662 (1988). 44j. W. Gordon, G. A. Scangos, D. J. Plotkin, J. A. Barbosa, and F. H. Ruddle, Proc. NatL Acad. Sci. U.S.A. 77, 7380 (1980).




transfection as well as control of the copy number of integrated sequences are required. Stable integration frequencies as high as 1 in 5 injected cells have been reported. In addition, high transfection efficiencies can be obtained even when as few as 5 copies of DNA are introduced per cell. 42 It has been reported that calcium phosphate-mediated transfection introduces an average of 1000 kb of DNA per recipient cell. 45 Though DNA microinjection has been used by a limited number of investigators, the advent of automated systems which permit injection of a large number of cells promises a wider use. Laser Method A sophisticated technology for the introduction of DNA into cultured cells has been described by Kurata e t aL ~s In this method, the DNA to be introduced into cells is dissolved in the culture medium surrounding the cells. Uptake of DNA by the cells is mediated by the introduction of minute holes in the cell membrane by brief pulses with a finely focused laser. Cells treated in such a manner repair the holes in their membrane within a fraction of a second, but nevertheless succeed in taking up DNA. Stable transfection efficiencies depend on the concentration of DNA in the culture medium and have been observed to be as high as 0.6%. The experimental apparatus is at least as complex as that required for microinjection, but allows for the more efficient treatment of a larger number of cells. Microprojectile-Mediated G e n e T r a n s f e r A novel method for gene transfer into maize cells has been reported 47 which utilizes a ballistic approach for introducing DNA into cells. In this procedure, DNA is adsorbed to microscopic tungsten particles in the presence of calcium chloride and spermidine. The DNA-adsorbed tungsten particles are then placed on the front surface of a cylindrical polyethylene macroprojectile, which is placed in the barrel of a particle gun device48 which is fired by a blank gunpowder charge. The barrel of the gun is directed toward suspension cells of maize on the bottom of a standard tissue culture dish. When the gun is fired, the macroprojectile moves down the barrel where it is impeded by a stopping plate. The microprojectiles 4s M. Perucho, D. Hanahan,and M. Wiglet,Cell 22, 309 (1980). S. Kurata,M. Tsukakoshi,T. Kasuya,and Y. Ikawa,Exp. CellRes. 162, 372 (1986). 47T. M. Klein,M. Fromm,A. Weissinger,D. Tomes,S. Schaff,M. Sletten,and J. C. Sanford, Proc. Natl. Acad. Sci. U.S.A. 85, 4305 (1988). 4sj. C. Sanford,T. M. Klein,E. D. Wolf,and N. Allen,Part. Sci. Technol. 5, 27 (1987).




continue past the stopping plate, penetrating the cells. When such a system was used for transfecting maize cells with an expressable chloramphenicol acetyltransferase (CAT) gene, CAT activity up to 200-fold over background was observed 24-96 hr after bombardment. Repeated bombardments of the same cell samples resulted in a proportionate increase in the observed levels of CAT activity. The major advantages of this system for gene transfer into maize cells are elimination of the need to generate protoplasts prior to transfection and elimination of the subsequent difficulty of generating whole plants from transfected protoplasts. The applicability of this method to mammalian cells has not been determined. Factors believed to be important to the success of the method include the size of the microprojectile, the velocity at which the microprojectiles are delivered, and the atmospheric pressure under which the bombardment takes place.

[42] S e l e c t i o n a n d C o a m p l i f i c a t i o n o f H e t e r o l o g o u s G e n e s in M a m m a l i a n C e l l s B y R A N D A L J. K A U F M A N

Introduction In the early 1950s, investigators established cell culture systems to study the mechanism by which cancer cells become resistant to a variety of chemotherapeutic agents, such as methotrexate (MTX). The initial investigations led to the observation that stepwise selection for growth of cultured animal cells in progressively increasing concentrations of MTX results in cells with increased levels of the target enzyme, dihydrofolate reductase (DHFR), as a consequence of a proportional increase in the DHFR gene copy number.] Subsequently, it has been observed that gene amplification is ubiquitous in nature and many, if not all, genes become amplified at some frequency (approximately l/104, although this number can vary extensively) (for recent review, see Ref. 2). With appropriate selection conditions, where the growth of cells harboring amplification of a particular gene is favored, a population of cells that contain the amplified gene will outgrow the general population. In the absence of drug selection, the amplified gene is most frequently lost. Since the degree of gene amplification, in most cases, is proportional to i F. W. Alt, R. E. Kellems, J. R. Bertino, a n d R. T. Schimke, J. Biol. Chem. 253, 1357 (1978). 2 R. T. Schimke, J. Biol. Chem. 263, 5989 (1988).


Copyright© 1990by AcademicPress,Inc. All rightsof reproduction in any form ~'served.

Methods for introducing DNA into mammalian cells.

[41] INTRODUCING DNA INTO MAMMALIANCELLS 527 experimentalist to retain authentic biological regulation of a recombinant cistron postintegration. Wh...
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