Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

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Gene Expression in Transfected Cells 355 27 Gene Expression in Transfected Cells Kate Hughes, Juha Saarikettu, and Thomas Grundström 1. Introduction A general approach to address the biological function of a calcium-binding protein, or another protein, in living cells is to increase or decrease the activity of the protein in the cell and analyze the effects on cell functions. In many cases, it is desirable to determine the effects of overexpressing the protein or a constitutively active or dominantly negative derivative, or to express the protein in a cell that normally lacks it. This is achieved by introducing its gene exogenously. The cDNA for the protein is cloned downstream of an active promoter in a plasmid designed for expression in mammalian cells. This expression plasmid is then transfected into the cell. Mammalian cells can be transfected by a number of methods. Among the more common techniques used today are cellular uptake of DNA prompted by chemical means such as cationic liposomes or DEAE-dextran, and the physical method of electroporation. These methods are relatively easy, and result in transfection of a large proportion of the cell population. Transfection mediated by chemical means rely on the responsiveness of the cell to the reagent used, and so the efficiency varies dramatically between cell types. Electroporation, however, is successful for a diversity of cell types, including many that are resistant to other methods of transfection. Furthermore, the lack of chemicals in the electroporation procedure reduces the risk of side effects on the cells. Liposome and DEAE-dextran transfection reagents can be purchased as kits with accompanying protocols from commercial sources such as Gene Therapy Systems, Invitrogen, Life Technologies, and Promega, and general overviews of these and other transfection procedures can be found in refs. 1–3. In this chapter, we will focus on electroporation. From: Methods in Molecular Biology, vol. 173: Calcium-Binding Protein Protocols, Vol. 2: Methods and Techniques Edited by: H. J. Vogel © Humana Press Inc., Totowa, NJ 355
356 Hughes et al. The electroporation procedure entails mixing cells with DNA and subjecting them to a high-voltage electric field, which transiently permeabilises the cell membrane allowing DNA to enter the cell (reviewed in refs. 4 and 5). Once inside the cell, the DNA is transported to the nucleus and transcription is initiated from the promoter of the expression plasmid. The choice of promoter is important in that it should enable a high level of transcription of the cDNA in the chosen cell type. Viruses such as the human cytomegalovirus (CMV), simian virus 40 (SV40), and Rous sarcoma virus (RSV) have naturally evolved strong promoters that function in a variety of cell types and are often the best choice. The level of protein expression also depends on a number of other factors, such as efficiency of transfection, cell type, and regulation of the protein by post-transcriptional mechanisms. For example, calmodulin is efficiently regulated post-transcriptionally (6–8), warning that profound increases in mRNA levels by transfection will not necessarily result in correspondingly profound increases in protein levels. To monitor the success of a transfection, it is common to include an internal control plasmid. In analogy to the expression construct, this plasmid contains the gene for an enzyme cloned downstream of a constitutively active promoter. At an appropriate time after transfection, a sample of cells is lysed and assayed for enzyme activity to ensure that the DNA was successfully delivered into the cells. By comparing the enzyme activity in the same number of cells from different transfections, it is also possible to normalize and thereby compare different transfections. It is far beyond the scope of this chapter to discuss the many possible analyses of functional consequences of (over-) expressing a calcium-binding protein or a protein regulated by a calcium-binding protein in living cells. However, one general process that can be readily assayed is the effect of the transfected protein on transcription from a gene control region. The regulatory sequence of interest is cloned into a plasmid upstream of a gene for a reporter enzyme. The chosen enzyme should be absent from mammalian cells and its activity easily measured. Luciferase is usually used for this purpose, as it can be measured by a convenient and very sensitive assay, whereas a gene for another luciferase enzyme that can be assayed independently, β-galactosidase or chloramphenicol acetyl transferase (CAT) is used in the internal control plasmid. The amount of reporter enzyme produced, determined by measuring its activity in a cell lysate, is proportional to the amount of transcription initiated from the regulatory sequence. The appeal of this simple method is that it also allows mutational analysis to define regions of a regulatory element that are important for the studied transcriptional regulation, as well as analyses of effects through individual transcription factors by using isolated specific DNA binding sites. A standard protocol for transient transfection of suspension cells by electroporation and analysis of the expression of reporter genes is described
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Methods in Molecular BiologyTM Biol
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M E T H O D S I N M O L E C U L A R
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© 2002 Humana Press Inc. 999 River
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Preface Calcium plays an important
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Contents Dedication ...............
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Contents xi 26 Enzymatic Assays to
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xiv Contents of Companion Volume 15
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xvi Contributors LESLIE D. HICKS
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20 Dean, Kelsey, and Re
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2 Yazawa
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4 Yazawa Fig. 1. Schematic represen
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6 Yazawa Fig. 2. Shop drawing for t
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8 Yazawa DuPont-NEN and the molar c
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10 Yazawa Fig. 4. Examples of the C
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12 Yazawa 5. Plot the calculated Ca
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14 Yazawa 12. Starovasnik, M. A., D
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16 Fig. 1. Molecular structures and
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18 Linse 7. 0.1 M EDTA. Dissolve 37
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20 Linse iterate the other paramete
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22 Linse tive to small alterations
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24 Linse References 1. Tsien, R. Y.
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26 Haiech and Kilhoffer to use the
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28 Haiech and Kilhoffer where γ is
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30 Haiech and Kilhoffer reporter gr
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32 Haiech and Kilhoffer tein with i
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34 Haiech and Kilhoffer Calmodulin
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36 Haiech and Kilhoffer Fig. 3. Mec
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38 Haiech and Kilhoffer We may sugg
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40 Haiech and Kilhoffer 29. Stewart
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42 Haiech and Kilhoffer 62. Becking
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44 Martin and Bayley Fig. 1. Absorp
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46 Martin and Bayley evaporation. C
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48 Martin and Bayley 4. Set the tem
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50 Martin and Bayley Fig. 2. Near-U
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52 Martin and Bayley discussed by M
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54 Martin and Bayley References 1.
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20 Dean, Kelsey, and Reik
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58 Fabian and Vogel are equipped wi
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60 Fabian and Vogel The penetration
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62 Fabian and Vogel perature, ionic
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64 Fabian and Vogel Fig. 3. IR spec
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66 Fabian and Vogel Fig. 4. Lower t
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68 Fabian and Vogel These correlati
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70 Fabian and Vogel Fig. 5. Amide I
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72 Fabian and Vogel 4. ATR-FTIR spe
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74 Fabian and Vogel 25. Georg, H.,
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76 Weljie and Vogel Fig. 1. Simplif
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78 Weljie and Vogel Fig. 3. Steady-
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80 Weljie and Vogel VIS spectrophot
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82 Weljie and Vogel 4. A series of
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84 Table 1 Advanced Fluorescence Me
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86 Weljie and Vogel References 1. L
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90 Johnson and Tikunova is removed
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92 Johnson and Tikunova function of
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94 Johnson and Tikunova Fig. 2. Rat
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96 Johnson and Tikunova Fig. 3. Rat
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98 Johnson and Tikunova Fig. 4. Ca
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100 Johnson and Tikunova cence inte
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102 Johnson and Tikunova 6. Johnson
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104 Julenius Fig. 1. Under conditio
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106 Julenius 3. Mix 50 µL of NHS s
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108 Julenius Fig. 3. A typical sens
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110 Julenius (2), is used in the Ia
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114 Lopez and Makhatadze Fig. 1. Th
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116 Lopez and Makhatadze 6. Buffers
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118 Lopez and Makhatadze The excess
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122 Lopez and Makhatadze Fig. 1. Is
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124 Lopez and Makhatadze 2.5 mL are
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126 Lopez and Makhatadze pendence o
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128 Hicks et al. equipped with a pu
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130 Hicks et al. Fig. 2. Debye plot
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132 Hicks et al. and 1.0 mg/mL for
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134 Hicks et al. Fig. 3. Sedimentat
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136 Hicks et al. 5. Hayes, D. B. (M
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138 Trewhella and Krueger This chap
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140 Trewhella and Krueger of the sc
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142 Trewhella and Krueger Table 1 C
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144 Trewhella and Krueger beam, or
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146 Trewhella and Krueger At very l
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148 Trewhella and Krueger analytica
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150 Trewhella and Krueger collapsed
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152 Trewhella and Krueger P(r) func
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154 Trewhella and Krueger Fig. 3. S
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156 Trewhella and Krueger concentra
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158 Trewhella and Krueger by a Nati
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162 Doherty-Kirby and Lajoie metal
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164 Doherty-Kirby and Lajoie two Ca
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166 Doherty-Kirby and Lajoie 7. Pep
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168 Doherty-Kirby and Lajoie Fig. 1
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170 Doherty-Kirby and Lajoie Fig. 2
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172 Doherty-Kirby and Lajoie 5. Alt
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174 Doherty-Kirby and Lajoie phosph
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176 Shaw Fig. 1. Ribbon drawing of
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178 Shaw 2.3. Other 1. Chemicals fo
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180 Shaw 3.3.3. Purification 1. Con
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182 Shaw 10. Hodges, R. S., Semchuk
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184 Brokx and Vogel Table 1 An Over
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186 Brokx and Vogel Fig. 1. UV abso
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188 Brokx and Vogel Fig. 2. 20% SDS
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190 Brokx and Vogel it through an a
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192 Brokx and Vogel 8. Weljie, A. M
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196 Berliner Fig. 1. Aqueous X-band
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198 Berliner in the Bruker instrume
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200 Berliner Fig. 3. ESR spectra of
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202 Berliner Fig. 5. Low-temperatur
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204 Berliner 14. Musci, G., Reed, G
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206 Clarke and Vogel cal calcium-bi
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208 Clarke and Vogel Fig. 1. 113 Cd
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214 Clarke and Vogel The linewidth
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218 Drakenberg sands, of Hertz broa
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220 Drakenberg 1. Bloch equations m
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222 Drakenberg Fig. 2. Measurement
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224 Drakenberg Fig. 3. (A) 43 Ca NM
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226 Drakenberg Fig. 5. 43 Ca NMR sp
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228 Drakenberg NMR at sub-mM concen
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230 Drakenberg 30. Shimizu, T., Hat
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232 Weljie and Heringa Table 1 Amin
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234 Weljie and Heringa Table 2 Webs
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236 Weljie and Heringa diction meth
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238 Weljie and Heringa these databa
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240 Weljie and Heringa lineages. Th
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242 Weljie and Heringa compared, an
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244 Weljie and Heringa sequences as
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246 Weljie and Heringa much larger
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248 Weljie and Heringa ment require
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250 Weljie and Heringa 10. Kawasaki
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252 Weljie and Heringa 44. Thompson
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254 Weljie and Heringa
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256 Li et al. In order for these ex
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258 Li et al. 7. Mineral Mixture (s
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260 Li et al. 3. When the 45% D 2O/
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262 Li et al. sion system works eff
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264 Li et al. reliably produce high
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268 Mal et al. NMR data, X-PLOR (11
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270 Mal et al. Table 1 Simulated An
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272 Mal et al. 3.1.2.1. AMBIGUOUS D
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274 Mal et al. molecular dynamics a
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276 Mal et al. Assuming a rigid bod
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278 Mal et al. assign (segid A and
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280 Mal et al. References 1. Drenth
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282 Mal et al. 33. Jeener, J., Meie
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286 Werner et al. Our research has
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288 Fig. 1. (A) T 1, T 2 and the he
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290 Werner et al. tion, using gradi
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292 Werner et al. Fig. 3. (A) Rotat
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294 Werner et al. Fig. 4. (A) Line-
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296 Werner et al. Table 2 Models of
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298 Werner et al. 9. Reinhardt, D.
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300 Werner et al. 39. Press, W. H.,
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302 Boyd et al. utilize residual di
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Page 331 and 332: 312 Boyd et al. 2. When studying mu
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Page 345 and 346: 326 Kobayashi that S-100A1 and S-10
Page 347 and 348: 328 Kobayashi 3.2. Coupling of Crom
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Page 353 and 354: 334 Kobayashi Both recombinant S-10
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Page 359 and 360: 340 Walsh et al. verts CaM from an
Page 361 and 362: 342 Walsh et al. 11. Bovine serum a
Page 363 and 364: 344 Walsh et al. Fig. 1. CaM-depend
Page 367 and 368: 348 Walsh et al. 3.5. NOS Reduction
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Page 377 and 378: 358 Hughes et al. 4. 1 M Na 2CO 3.
Page 379 and 380: 360 Hughes et al. range, repeat ste
Page 381 and 382: 362 Hughes et al. Table 1 Examples
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Page 385 and 386: 366 Persechini of the different CaM
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Page 389 and 390: 370 Persechini 2. 1 L of Terrific b
Page 391 and 392: 372 Persechini Fig. 4. Titration wi
Page 393 and 394: 374 Persechini Table 1 Parameters f
Page 395 and 396: 376 Persechini Fig. 6. The [Ca 2+ -
Page 397 and 398: 378 Persechini determined if the di
Page 399 and 400: 380 Persechini relatively insensiti
Page 401 and 402: 382 Persechini 18. Chafouleas, J. G
Page 403 and 404: 384 Török et al. actions in the c
Page 405 and 406: 386 Török et al. 3. The reaction
Page 407 and 408: 388 Török et al. Fig. 2. Electros
Page 409 and 410: 390 Török et al. Fig. 3. Peptides
Page 411 and 412: 392 Török et al. Table 1 Peptide
Page 413 and 414: 394 Török et al. Fig. 7. Nanospra
Page 415 and 416: 396 Török et al. Fig. 9. Nanospra
Page 417 and 418: 398 Török et al. Fig. 11. Electro
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Page 421 and 422: 402 Török et al. Fig. 14. Localiz
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406 Török et al. the significantl
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408 Török et al.
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410 Index substitutes, see Fluoresc
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412 Index Stern-Volmer plot, 78, 81
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414 Index Phenothiazines, see Calmo
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METHODS IN MOLECULAR BIOLOGY TM •
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