Gene Biotechnology

Gene Biotechnology......Page 1
Preface......Page 4
Authors......Page 6
Contents......Page 8
Introduction......Page 10
Contents......Page 0
Proposal 1. Identification Of New Drug-targeting Proteins And Isolation Of Novel Genes......Page 11
Proposal 5. Identification Of Toxicant-binding Proteins And Isolation Of A Novel Gene Related To The Toxicant And Heart Hypertrophy Using An Animal Model......Page 13
Proposal 7. Discovery Of Iaa- Or Ga 3 -binding Proteins And Isolation Of Novel Genes In Plants......Page 17
References......Page 22
Introduction......Page 24
Design And Synthesis Of Specific Forward And Reverse Primers......Page 25
Amplification Of Cdna Of Interest By Rt-pcr......Page 28
Purification Of Pcr Products By High-speed Centrifugation Of Agarose Gel Slices......Page 30
Amplification And Isolation Of Cdna Ends By 3 ¢ -race......Page 31
Partial Digestion Of Genomic Dna Using Sau3a I......Page 33
Design And Synthesis Of Specific Forward And Reverse Primers......Page 36
Amplification Of Specific Dna Fragments By Pcr......Page 37
References......Page 38
Contents......Page 39
Introduction......Page 40
Part A. Construction And Screening Of A Subtracted Cdna Library......Page 43
Isolation Of Total Rnas From Cell Or Tissue Types A And B Of Interest......Page 45
Protocol A. Rapid Isolation Of Total Rna By Acid Guanidinium Thiocyanate-phenol-chloroform Method......Page 46
Protocol B. Rapid Isolation Of Total Rnas Using Trizol Reagent Tm (gibco Brl Life Technologies)......Page 47
Protocol C. Measurement Of Rnas......Page 48
Protocol A. Purification Of Poly(a)+rnas Using Oligo(dt)-cellulose Column......Page 50
Protocol B. Minipurification Of Mrnas Using Oligo(dt)–cellulose Resin......Page 52
Protocol A. Synthesis Of First-strand Cdnas From Mrnas......Page 53
Protocol C. Analysis Of Cdnas By Alkaline Agarose Gel Electrophoresis......Page 55
Separation Of Cdna/mrna Hybrids From Single-stranded Cdnas By Hydroxyapatite (hap) Chromatography......Page 58
Synthesis Of Double-stranded Cdnas From Subtracted Cdnas......Page 59
Ligation Of Ecor I Linkers Or Adaptors To Double-stranded, Blunt-end Cdnas......Page 60
Ligation Of Cdnas To Vectors......Page 65
Titration Of Packaged Phage......Page 66
Amplification Of Cdna Library (optional)......Page 68
General Principles And Considerations Of An Expression Cdna Library......Page 69
Method A. Immunoscreening Of Expression Cdna Library Using Specific Antibodies......Page 71
Method D. Isolation Of L Phage Dnas By The Liquid Method......Page 76
Troubleshooting Guide......Page 78
References......Page 80
Introduction......Page 81
Selection Of Restriction Enzymes......Page 82
Protocols For Restriction Enzyme Digestion......Page 84
Elution Of Dna Bands By High-speed Centrifugation Of Agarose Gel Slices......Page 87
Elution Of Dna Fragment By Melting And Thawing Of Agarose Gel Slices2,3......Page 88
Elution Of Dna Fragment Using Na45 Deae Membrane......Page 89
Ligation Of Dna Fragments......Page 90
Single-step Cloning By Pcr......Page 91
Protocol 1. Preparation Of Competent Cells For Transformation......Page 93
Protocol 2. Transformation Of Cells By Cacl2 Method......Page 95
Protocol 3. Transformation By Electroporation......Page 96
Protocol 1. Minipreparation Of Plasmid Dna......Page 97
Protocol 2. Large-scale Preparation Of Plasmid Dna......Page 100
Protocol 3. Purification Of Plasmid Dna By Cscl Gradient......Page 101
Verification Of Dna Insertion By Restriction Enzyme Digestion And Agarose Gel Electrophoresis......Page 103
Troubleshooting Guide......Page 105
References......Page 106
Contents......Page 107
Introduction......Page 108
Purification Of Double-stranded Plasmid Dna By The Alkaline Method......Page 110
Purification Of Single-stranded Dna......Page 113
Method A. Sequencing Reactions For Double-stranded Plasmid Dna......Page 114
Protocol 3. Preparation Of Sequencing Gels......Page 119
Method A. Pouring The Gel Mixture Horizontally Into The Sandwich......Page 121
Method B. Pouring The Gel At An Angle......Page 122
Protocol 4. Electrophoresis......Page 124
Protocol 5. Transferring Dna From Gel Onto A Nylon Membrane......Page 126
Method A. Chemiluminescent Detection......Page 127
Method B. Colorimetric Detection......Page 128
Isotopic Dna Sequencing Method......Page 130
The Use Of Formamide Gels......Page 131
Dna Sequencing By Primer Walking......Page 132
Dna Sequencing By Unidirectional Deletions......Page 133
Protocol 7. Performing A Series Of Deletions Of The Linearized Dna With Exonuclease Iii And Recircularization Of Dna With T4 Dna Ligase (figure 5.3)......Page 135
Preparation Of Pcr And Sequencing Reactions......Page 137
Rna Sequencing......Page 139
Protocol 3. Termination Reaction......Page 140
Troubleshooting Guide......Page 142
References......Page 144
Contents......Page 146
Submission Of A Sequence To Genbank......Page 147
Blastn......Page 149
Blastx......Page 152
Blastp......Page 153
Sequence Entry......Page 154
Combination Or Assembly Of Multiple Fragments Into A Single Sequence......Page 156
Enter Sequences To Be Assembled Into The Project File Generated In A (e.g., Fragment) Using The Gelenter Program......Page 157
Compare And Identify Overlap Points Of Entered Fragments Using The Gelmerge Or Geloverlap Program......Page 158
Assemble And Review The Combined Sequence By Using The Gelassemble Program......Page 159
Exhibition Of Restriction Enzymes Above Both Strands Of A Dna Sequence And Possible Protein Translation Below The Sequence Using The Map Program......Page 160
Identification Of Specific Restriction Enzyme Cutting Sites And Sizes Of Fragments By Using The Mapsort Program......Page 162
Comparison Of Similarity Between Two Sequences......Page 163
Translate......Page 165
Backtranslate (using The Sequence1.pep As An Example)......Page 166
Identification Of Enzyme Digestion Sites Within A Peptide Or Protein......Page 167
Obtaining Nucleotide And Amino Acid Sequences From Genbank......Page 168
References......Page 169
Introduction......Page 171
Principles And General Considerations......Page 172
Restriction Enzyme Digestion Of Dnas......Page 173
Agarose Gel Electrophoresis Of Dnas......Page 174
Method A. Upward Capillary Transfer (6 To 12 H)......Page 175
Method B. Downward Capillary Transfer (1 To 1.5 H Using Alkaline Buffer Or 3 H Using Neutral Buffer)......Page 178
Preparation Of Nonisotopic Dna Probes......Page 180
Preparation Of Isotopic Dna Probes......Page 184
Prehybridization And Hybridization......Page 188
Protocol B. Washing And Antibody Incubation Of Filters Hybridized With Biotin-dutp- Or Dig-utp-labeled Probes......Page 189
Method A. Chemiluminescent Detection......Page 190
Method C. Detection Of Signals By Autoradiography......Page 191
Troubleshooting Guide......Page 196
References......Page 197
Contents......Page 198
Introduction......Page 199
Design And Selection Of Plasmid-based Expression Vectors......Page 200
Selectable Marker Genes......Page 201
Splicing Regions......Page 202
Retrovirus Vectors......Page 203
Preparation Of Plasmid Sense Cdna Constructs......Page 207
Method A. Transfection By Calcium Phosphate Precipitation......Page 208
Protocol 1. Preparation Of Viral Supernatant By Transient Transfection Of A Packaging Cell Line With Retrovirus Vector Constructs......Page 210
Protocol 3. Determination Of Viral Titer......Page 212
Protocol 4. Amplification Of Virus Stock By Serial Reinfection Of Fresh Target Cells......Page 213
Protocol 5. Transfection Of Cells Of Interest With High-titer Stock Of Replication-incompetent Retroviruses (figure 8.9)......Page 215
Method B. Transfection By Electroporation......Page 216
Hygromycin- B -phosphotransferase......Page 217
Characterization Of Stably Transfected Cell Clones......Page 218
Isolation Of Genomic Dna From Cultured Cells......Page 219
Analysis Of Southern Blot Data......Page 220
Activity Assay Of Chloramphenical Acetyl Transferase (cat)......Page 221
Luciferase Assay......Page 222
B -galactosidase Staining Of Cells......Page 223
Troubleshooting Guide......Page 225
References......Page 226
Contents......Page 228
Design And Synthesis Of Antisense Oligonucleotides......Page 229
Treatment Of Cultured C Ells With Antisense Oligomers And Determination Of O Ptimum Dose Of Oligomers By Western Blot Analysis......Page 231
Treatment Of Cultured Cells Using An Optimum Dose Of Oligomers......Page 232
Design And Selection Of Plasmid-based Expression Vectors......Page 233
Transient Transfection Of Cultured Cells With Antisense Constructs......Page 234
Method A. Transfection By Liposomes......Page 235
Method B. Transfection By Microinjection......Page 236
Selection Of Stably Transfected Cell Lines With Appropriate Drugs......Page 237
Examination Of Expression Of Antisense Rna By Northern Blotting......Page 238
Generation Of Transgenic Mice......Page 239
Method A. Production Of Transgenic Mice From Stably Transfected Es Cells......Page 240
Method B. Production Of Transgenic Mice From Oocytes......Page 246
Characterization Of Transgenic Mice......Page 248
Troubleshooting Guide......Page 249
References......Page 250
Introduction......Page 251
Principles And General Considerations......Page 252
Electrophoresis Of Rnas Using Formaldehyde Agarose Gels......Page 254
Protocol B. Preparation Of Rna Probes By Transcription In Vitro......Page 257
Analysis Of Mrna Expression By A Semiquantitative Pcr Approach......Page 261
Troubleshooting Guide......Page 265
References......Page 266
Introduction......Page 268
Principles......Page 270
Protocol 1. Protein Extraction From Cultured Cells......Page 274
Protocol 1. Preparation Of The Sds Separation Gels......Page 275
Protocol 2. Preparation Of Stacking Gels......Page 276
Protocol 3. Electrophoresis......Page 277
Protocol 4. Staining And Destaining Of Sds-page Using A Modified Coomassie Blue (cb) Method (figure 11.2)......Page 278
Protocol 5. Transfer Of Proteins From Sds-page Onto A Nitrocellulose Or Pvdf Membrane By Electroblotting (figure 11.3)......Page 279
Protocol 6. Blocking And Hybridization Of The Membrane Filter Using Specific Antibodies......Page 281
Protocol 7. Detection Of Hybridized Signals......Page 282
Analysis Of Proteins By 2-d Gel Electrophoresis......Page 283
Troubleshooting Guide......Page 286
References......Page 287
Contents......Page 289
Preparation Of Silane-coated Glass Slides......Page 290
Preparation Of 4% (w/v) Paraformaldehyde (pfa) Fixative (1 L)......Page 291
Fixation......Page 292
Embedding......Page 293
Cryosectioning......Page 294
Fixation......Page 295
Dewaxing Of Sections......Page 296
Protease Digestion......Page 297
Rnase Treatment For In Situ Hybridization......Page 298
Synthesis Of Probes For Dna Hybridization Using Random Primer Labeling Of Dsdna......Page 299
Preparation Of [35s]utp Riboprobe For Rna Hybridization By Transcription In Vitro Labeling......Page 302
In Situ Hybridization......Page 304
Detection Of Hybridized Signals......Page 306
References......Page 310
Introduction......Page 312
Method A. Random Primer Digoxigenin Labeling Of Ds Dna......Page 313
Method B. Nick Translation Labeling Of Dsdna With Biotin -11-dutp Or Digoxigenin-11-dutp......Page 314
Method C. Riboprobe Or Rna Labeling......Page 315
In Situ Hybridization Of Specimens Fold. By Crc Press Llc......Page 318
Enzymatic Detection Of Hybridized Signals Using Colorimetric Substrates Nbt And Bcip......Page 319
Fluorescence Detection Of Hybridized Signals......Page 320
Troubleshooting Guide......Page 322
References......Page 323
Contents......Page 324
Introduction......Page 325
Part A. Detection Of A Low Copy Gene By In Situ Pcr Hybridization......Page 326
Design Of Specific Primers For In Situ Pcr Amplification Of Target Dna Sequences......Page 327
Carrying Out Fixation Of Cells Or Tissues, Tissue Embedding, Sectioning And Mounting Of Sections On Slides......Page 329
Performance Of In Situ Pcr......Page 330
Performance Of In Situ Hybridization And Detection By Rish Or By Fish......Page 333
Carrying Out Cell Or Tissue Fixation, Tissue Embedding, Sectioning And Mounting Of Sections On Slides......Page 334
Performance Of In Situ Reverse Transcription (isrt) Reaction On The Sections......Page 336
Performance Of In Situ Hybridization And Detection By Rish Or By Fish......Page 337
Troubleshooting Guide......Page 338
References......Page 339
Contents......Page 341
Selection Of Lambda Vectors......Page 342
Determination Of The Optimal Partial Digestion Of Genomic Dna With Sau 3a I......Page 344
Large -scale Preparation Of Partial Digestion Of Genomic Dna......Page 346
Partial Fill-in Of Recessed 3' Termini Of Genomic Dna Fragments......Page 347
Small-scale Ligation Of Partially Filled -in Genomic Dna Fragments And Partially Filled-in Ygem-12 Arms......Page 348
Titration Of Packaged Phage On Lb Plates......Page 349
Method A. Screening Of The Genomic Library With A A-32p-labeled Probe......Page 351
Method B. Screening Of A Genomic Library Using A Nonisotopelabeled Probe......Page 356
Isolation Of Lphage Dnas By Liquid Method......Page 358
Restriction Mapping Of Recombinant Bacteriophage Dna Containing Genomic Dna Insert Of Interest......Page 360
Troubleshooting Guide......Page 362
References......Page 363
Introduction......Page 364
Protocol 1. Culture Of Es Cells In Media Containing Leukemia Inhibitory Factor (lif)......Page 365
Protocol 2. Preparation Of Mitotically Inactivated Sto Cells As Feeder Layers For Growth Of Es Cells......Page 366
Protocol 3. Culture Of Es Cells On Fibroblast Feeder Layers......Page 367
Trypsinizing......Page 368
Freeze Back......Page 369
Protocol 5. Cell Counting And Determination Of Cell Density......Page 370
Troubleshooting Guide......Page 372
References......Page 373
Contents......Page 374
Introduction......Page 375
Overview And General Principles Of Novel Strategies: Gene Double-knockout And Temporal Silence......Page 376
Protocol B. Deletion Of Exons Of A Specific Gene......Page 379
Protocol C. Preparation Of Isogenic Dna By Pcr......Page 381
Type A. Neor Gene Vector For First Copy Of Gene Knockout......Page 387
Type B. Hygr Gene Vector For The Second Copy Of Gene Knockout......Page 388
Type C. Hyrtr Gene Vector For Keeping The Double-knockout Gene Silent......Page 389
Method A. Transfection By Electroporation......Page 390
Method B. Transfection Of Cells By Lipofectin Reagents......Page 394
Double Selection Of Cells Containing A Double-knockout Gene......Page 395
Triple Selections Of Cells Containing A Double-knockout Gene......Page 396
B-galactosidase Staining Of Cells......Page 397
Southern Blot Analysis......Page 398
Western Blot Analysis......Page 402
Procedure 2. Preparation Of A Bank Of Sterile Males By Vasectomy......Page 404
References......Page 405
Contents......Page 407
Part A. Expression And Purification Of Proteins In Prokaryotic Systems......Page 408
Design And Selection Of Bacterial Expression Vectors......Page 409
Cloning Cdna Inserts Into Vectors......Page 411
Protocol B. Induction Of Protein Expression With Iptg......Page 413
Protocol D. Induction Of Protein Expression By Heat Shock......Page 415
Extraction Of Total Proteins For Purification......Page 416
Rapid Purification Of The Expressed Proteins With His-tag Affinity Column Chromatography......Page 418
Protease Cleavage Of The His-tag......Page 419
Protocol 1. Enzymatic Cleavage Of The Leader Sequence By Factor Xa......Page 420
Protocol 2. Enzymatic Cleavage Of The Leader Sequence With Thrombin......Page 422
Part B. Overexpression Of Specific Proteins In Eukaryotic Systems......Page 423
Cloning A Cdna To Be Expressed Into Expression Vectors......Page 425
Protocol A. Linearization Of Plasmid Dna......Page 427
Protocol B. Transformation Of Pichia By Electroporation......Page 428
Protocol C. Preparation Of Spheroplasts For Transformation......Page 430
Protocol D. Transformation Of Spheroplasts With Linearized Dna......Page 433
Screening And Isolation Of His+mut+ And His+muts Phenotype......Page 435
Determination Of Optimum Conditions For Protein Expression......Page 438
Extraction Of Total Proteins For Purification......Page 440
References......Page 441
Introduction......Page 443
Isolation Of Total Rna From Tissues Or Organs Of Interest......Page 446
Isolation Of Total Rna From Cultured Cells......Page 447
Designing Primers And Probes......Page 449
Assembly Of Reactions......Page 450
Analysis Of Data......Page 452
Validation......Page 453
References......Page 456
INTRODUCTION......Page 458
ISOLATION OF TOTAL RNA FROM CELLS OR TISSUES......Page 459
PREPARATION OF FLUORESCENT PROBES FROM mRNA......Page 460
GENERATION OF DNA MICROARRAYS OR CHIPS......Page 462
WASHING......Page 463
SCANNING GENE CHIPS USING GENEPIX 4000A MICROARRAY SCANNER......Page 464
REFERENCES......Page 467
INTRODUCTION......Page 468
Preparation of Lymphocytes from Tissues......Page 471
Isolation of Total RNA from Lymphocytes......Page 472
Synthesis of First-Strand cDNAs from mRNAs......Page 473
Synthesis of Double-Stranded cDNAs from the Subtracted cDNAs......Page 476
PCR Amplification of Heavy- and Light-Chain Variable Regions (VH, VL), Linker and Assembly of Recombinant Single-Chain DNA via PCR......Page 477
Infection of Bacteria Carrying Recombinant DNA Constructs and Rescuing Phagemid Libraries......Page 484
Selection of Phage–Antibody Particles by Biopanning......Page 487
References......Page 488
INTRODUCTION......Page 489
Selection of siRNA Target Site......Page 490
Preparation of Double-Stranded Oligonucleotide Fragment for Cloning......Page 492
Cloning Hairpin siRNA Oligo Insert into Appropriate Vectors......Page 493
Purification of Plasmid DNA Carrying Hairpin siRNA Insert......Page 495
Transient Transfection of Cells of Interest with Purified Plasmid DNA to Express siRNAs for Inhibiting Target mRNA......Page 497
Isolation of Total RNA from Transfected Cells......Page 499
Analysis of Inhibition of Specific mRNA via siRNAs by Northern Blotting......Page 500
Extraction of Proteins for Reporter Activity Assays......Page 501
Reporter Gene Assays of Down-Regulation of Gene Expression via siRNAs......Page 502
References......Page 503