 |
Gene biotechnology / by William Wu ... [et al.]. — 3rd ed. — Boca Raton : CRC Press, c2011. – (58.21/G326f/3rd ed.) |
Contents
Contents
Preface xxvii
Authors xxix
Chapter 1 Strategies for Novel Research Projects and/or Research Grant Funding 1
1.1 Introduction 1
References 14
Chapter 2 Rapid Isolation of Specific cDNAs or Genes by PCR 17
2.1 Introduction 17
2.2 Isolation of Specific Full-Length cDNAs by RT-PCR Method 17
2.3 Amplification and Isolation of cDNA Ends by 5'-RACE 23
2.4 Amplification and Isolation of cDNA Ends by 3'-RACE 24
2.5 Isolation of Gene of Interest by PCR 24
2.6 Subcloning of cDNA or Gene of Interest 30
2.7 Characterization of PCR Products 30
References 30
Chapter 3 Construction and Screening of Subtracted and Complete Expression cDNA Libraries 33
3.1 Introduction 33
3.2 Construction and Screening of a Subtracted cDNA Library 36
3.3 Construction and Screening of a Complete Expression cDNA Library 61
Chapter 4 Subcloning of Genes or DNA Fragments 73
4.1 Introduction 73
4.2 Restriction Enzyme Digestion of Vector or DNA Insert for Subcloning 73
4.3 Purification of DNA Fragments from Agarose Gels 78
4.4 Ligation of DNA Fragments 81
4.5 Single-Step Cloning by PCR 83
4.6 Transformation of Ligated DNA into Bacteria 85
4.7 Isolation and Purification of Plasmid DNA by Alkaline Method 88
4.8 Verification of DNA Insertion by Restriction Enzyme Digestion and Agarose Gel Electrophoresis 94
4.9 Verification of Insertion Site by DNA Sequencing 95
References 96
Chapter 5 Nonisotopic and Isotopic DNA or RNA Sequencing 97
5.1 Introduction 97
5.2 Nonisotopic DNA Sequencing Method 99
5.3 Isotopic DNA Sequencing Method 117
5.4 Use of Formamide Gels 119
5.5 Extending Sequencing Far from the Primers 120
5.6 DNA Sequencing by Primer Walking 120
5.7 DNA Sequencing by Unidirectional Deletions 120
5.8 Direct DNA Sequencing by PCR 124
5.9 RNA Sequencing 127
References 132
Chapter 6 Bioinformation Superhighway and Computer Databases of Nucleic Acids and Proteins 133
6.1 Introduction 133
6.2 Communication with GenBank via the Internet 133
6.3 Computer Analysis of DNA Sequences by the GCG Program 140
References 153
Chapter 7 Characterization of DNA or Genes by Southern Blot Hybridization 155
7.1 Introduction 155
7.2 Principles and General Considerations 155
7.3 Isolation of DNA for Analysis 157
7.4 Restriction Enzyme Digestion of DNA 157
7.5 Agarose Gel Electrophoresis of DNAs 157
7.6 Blotting of DNAs onto Nylon Membranes 159
7.7 Preparation of Probes 163
7.8 Prehybridization and Hybridization
7.9 Washing and/or Incubation of Antibodies
7.10 Detection of Hybridized Signal(s)
References
Chapter 8 Gene Overexpression by Sense RNA in Mammalian Systems 181
8.1 Introduction 181
8.2 Design and Selection of Plasmid-Based Expression Vectors 183
8.3 Preparation of Plasmid Sense cDNA Constructs 190
8.4 Transient Transfection of Mammalian Cells with Sense Constructs 190
8.5 Stable Transfection of Mammalian Cells with Sense DNA Constructs 198
8.6 Selection of Stably Transfected Cell Lines with Appropriate Drugs 199
8.7 Characterization of Stably Transfected Cell Clones 200
8.8 Generation of Transgenic Mice from Sense ES Clones 207
8.9 Characterization of Transgenic Mice 207
References 208
Chapter 9 Gene Underexpression in Cultured Cells and Animals by Antisense DNA and RNA Strategies 209
9.1 Introduction 209
9.2 Antisense Oligonucteotide Approaches 209
9.3 Design and Selection of Plasmid-Based Expression Vectors 214
9.4 Preparation of Plasmid Antisense cDNA Constructs 214
9.5 Transient Transfection of Cultured Cells with Antisense Constructs 215
9.6 Stable Transfection of Cultured Cells with Antisense DNA Constructs 215
9.7 Selection of Stably Transfected Cell Lines with Appropriate Drugs 218
9.8 Characterization of Stably Transfected Cell Clones 218
9.9 Generation of Transgenic Mice 220
9.10 Characterization of Transgenic Mice 228
References 229
Chapter 10 Analysis of Oene Expression at Functional Genomic Level Using Northern Blotting or PCR 231
10.1 Introduction
10.2 Principles and General Considerations
10.3 Isolation of Total RNAs and/or Purification of mRNAs..
10.4 Electrophoresis of RNAs Using Formaldehyde Agarose Gels
10.5 Blotting of RNAs onto Nylon Membranes by the Capillary Method
10.6 Preparation of Isotopic or Nonisotopic DNA/RNA Probes
10.7 Hybridization and Detection of Signals
10.8 Analysis of mRNA Expression by a Semiquantitative PCR Approach
References
Chapter 11 Analysis of Gene Expression at Proteomic Level via Western Blotting 249
11.1 Introduction 249
11.2 Principles 251
11.3 Extraction of Cellular Proteins 253
11.4 Analysis of Proteins by SDS-PAGE and Western Blotting... 255
11.5 Analysis of Proteins by 2-D Gel Electrophoresis 263
References 266
Chapter 12 Analysis of Cellular DNA or Abundance of mRNA by Radioactivity in Situ Hybridization 269
12.1 Introduction 269
12.2 Tissue Fixation， Embedding， Sectioning， and Mounting of Sections on Slides
12.3 In Situ Hybridization and Detection Using Isotopic Probes 275
References 289
Chapter 13 Localization of DNA or Abundance of mRNA by Fluorescence in Situ Hybridization 291
13.1 Introduction 291
13.2 Cell or Tissue Fixation， Tissue Embedding， Sectioning， and Mounting 291
13.3 Dewaxing of Sections， Protease Digestion， and DNase or RNase Treatment of Specimens for in Situ Hybridization 292
13.4 Preparation of Nonisotopic Probes Using One of the Following Methods， Depending on Specific Probe and Detection Strategies 292
13.5 In Situ Hybridization of Specimens 296
13.6 Enzymatic Detection of Hybridized Signals Using Colorimetric Substrates NBT and BCIP 297
13.7 Fluorescence Detection of Hybridized Signals 298
References 301
Chapter 14 In Situ PCR Hybridization of Low Copy Genes and in Situ RT-PCR Detection of Low Abundance mRNAs 303
14.1 Introduction 303
14.2 Detection of a Low Copy Gene by in Situ PCR Hybridization 304
14.3 Detection of Low Abundance mRNA by in Situ RT-PCR Hybridization 312
14.4 In Situ PCR or RT-PCR Detection of DNA or mRNA by Direct Incorporation of Digoxigenin- 11- dUTP or Biotin-dUTP without Hybridization 316
References 317
Chapter 15 Isolation and Characterization of Genes from Genomic DNA Libraries 319
15.1 Introduction 319
15.2 Selection of Lambda Vectors 320
15.3 Isolation of High Molecular Weight of Genomic DNA 320
15.4 Partial Digestion of Genomic DNA Using Sau3AI 322
15.5 Partial Fill-In of Recessed 3'-Termini of Genomic DNA Fragments 324
15.6 Ligation of DNA Inserts to Vectors 325
15.7 Screening of Genomic DNA Library and Isolation of Specific Clones 328
15.8 Isolation of X Phage DNAs by the Liquid Method 335
15.9 Restriction Mapping of Recombinant Bacteriophage DNA Containing the Genomic DNA Insert of Interest 336
15.10 Subcloning of the Isolated Gene or DNA Fragments 338
15.11 Characterization of the Isolated DNA 338
References 339
Chapter 16 Mouse Stem Cells as a Model Mammalian Cell Line for Gene Expression 341
16.1 Introduction 341
16.2 Protocol 1. Culture of ES Cells in Media Containing Leukemia Inhibitory Factor
16.3 Protocol 2. Preparation of Mitotically Inactivated STO Cells as Feeder Layers for Growth of ES Cells 343
16.4 Protocol 3. Culture of ES Cells on Fibroblast Feeder Layers 344
16.5 Protocol 4: Trypsinizing， Freezing， and Thawing of Mammalian Cells 344
16.6 Protocol 5. Cell Counting and Determination of Cell Density 347
References 349
Chapter 17 Strategies for Gene Double Knockout 351
17.1 Introduction 351
17.2 Overview and General Principles of Strategies: Gene Double-Knockout and Temporal Silence 352
17.3 Manipulation of the Specific Gene to Be Targeted 354
17.4 Design and Selection of Vectors and Marker Genes for Gene Double Knockout and Temporal Silence 362
17.5 Preparation of DNA Constructs for a Double Knockout and a Silencer 365
17.6 Transfection of ES Cells with Double Knockout and Silencer DNA Constructs
17.7 Selection and Characterization of Targeted ES Cell Clones 370
17.8 Characterization of Targeted ES Cell Clones 371
17.9 Generation of Transgenic Offspring from Double-Knockout ES Cells 377
17.10 Characterization of Mutant Mice 379
References 379
Chapter 18 Large-Scale Expression and Purification of Recombinant Proteins in Cultured Cells 381
18.1 Introduction 381
18.2 Expression and Purification of Proteins in Prokaryotic Systems 382
18.3 Overexpression of Specific Proteins in Eukaryotic Systems
References
Chapter 19 Quantitative Analysis of Functional Genome by Real-Time RT-PCR 415
19.1 Introduction 415
19.2 Isolation of Total RNA from Tissues or Organs of Interest 417
19.3 Isolation of Total RNA from Cultured Cells 418
19.4 Preparation of RNA Pool 419
19.5 Designing Primers and Probes 420
19.6 Real-Time RT-PCR 421
References 427
Chapter 20 High-Throughput Analysis of Gene Expression by Cutting-Edge Technology--DNA Microarrays (Gene Chips) 429
20.1 Introduction 429
20.2 Isolation of Total RNA from Cells or Tissues 430
20.3 Preparation of Fluorescent Probes from mRNA 431
20.4 Generation of DNA Microarrays or Chips 433
20.5 Prehybridization of Gene Chips or Arrays 433
20.6 Hybridization 434
20.7 Washing 434
20.8 Scanning of Gene Chips Using GenePix 4000a Microarray Scanner 435
References 437
Chapter 21 Construction and Screening of Human Antibody Libraries Using Phage Display Technology 439
21.1 Introduction 439
21.2 Preparation of White Blood Cells (WBCs) or Lymphocytes for RNA Isolation 441
21.3 Isolation of Total RNA from Lymphocytes 442
21.4 Synthesis of cDNAs 442
21.5 Design of Primers for Construction of Antibody V Gene Repertoire 446
21.6 PCR Amplification of Heavy- and Light-Chain Variable Regions (VH， VL)， Linker， and Assembly of Recombinant Single-Chain DNA via PCR 449
21.7 Generation of Recombinant DNA Constructs for Expressing Recombinant Antibodies on Surface of Phage
21.8 Expression， Biopanning， and Characterization of Phage Produces Monoclonal Antibodies on Its Tip 451
References 456
Chapter 22 Down-Regulation of Gene Expression in Mammalian Systems via siRNA Technology 457
22.1 Introduction
22.2 Design of Oligonucleotides for Hairpin siRNA
22.3 Preparation of Double-Stranded Oligonucleotide Fragment for Cloning
22.4 Cloning Hairpin siRNA Oligo Insert into Appropriate Vectors
22.5 Purification of Plasmid DNA Carrying Hairpin siRNA Insert 462
22.6 Transient Transfection of Cells of Interest with Purified Plasmid DNA to Express siRNAs for Inhibiting Target mRNA 463
22.7 Isolation of Total RNA from Transfected Cells 465
22.8 Analysis of Inhibition of Specific mRNA via siRNAs by Northern Blotting 466
22.9 Extraction of Proteins for Proteomic and/or Reporter Gene Analysis 466
22.10 Analysis of siRNA-Mediated Inhibition of Gene Expression at Proteomic Level by Western Blotting 467
22.11 Reporter Gene Assays of Down-Regulation of Gene Expression via siRNAs 467
References 468
Chapter 23 Strategies for Gene Cloning， Expression， and Identification of Protein-Protein Interaction 471
23.1 Introduction 471
23.2 Rapid and High-Efficiency PCR-TA Cloning without DNA Ligase 471
23.3 Transfection of Eukaryotic Cells for Expression of Proteins of Interest 475
23.4 Protein Extraction 476
23.5 Coimmunoprecipitation 477
23.6 Analysis and Identification of Binding Proteins 477
23.7 Different Strategies for Cloning， Identification， Mapping， and Characterization of Protein-Protein Interaction 479
23.8 Exploring the Biology or Potential Mechanisms of the Interactive Proteins
References 481
Chapter 24 Conditional Gene Knockout 483
24.1 Introduction 483
24.2 Generation of Cre-loxP DNA Constructs 484
24.3 Generation of Transgenic Mouse Line Containing loxP Cassette， Cre Cassette， or Both
24.4 Generation and Characterization of Stable Transgenic Mouse Lines A and B
24.5 Generation and Characterization of Transgenic Mouse Line C
References
Chapter 25 How to Write a Research Manuscript for Publication in an English Journal
25.1 Introduction 501
25.2 Title 503
25.3 Authors and Affiliation 503
25.4 Materials and Methods 504
25.5 Results 505
25.6 Abstract (Summary in Some Journals) 506
25.7 Introduction 506
25.8 Discussion 507
25.9 Acknowledgments 508
25.10 References 508
25.11 Adjustment of Format of Manuscript 508
25.12 Fine-Tuning and Submission of Manuscript 509
25.13 Some Observations on to Which Journal to Submit Your Manuscript 509
25.14 Waiting for Reviewers' Comments， Revision of Manuscript Based on Reviewers' Critique， and Resubmission of Manuscript 509
References 510
Chapter 26 How to Protect Your Discovery and Invention: Patent 101 511
26.1 Introduction 511
26.2 Research Discoveries vs. IPs， Invention 511
26.3 IP or Invention Documentation 512
26.4 Technology Disclosure 512
26.5 Provisional Application for Patent 513
26.6 Full-Length or Complete Patent Application 514
References 519
Chapter 27 Determination of Transgene Copy Numbers and Practical Biocalculation
27.1 Introduction 521
27.2 Determination of Gene Copy Numbers in Transgenic Animals 521
27.3 Molecular Weight 524
27.4 Calculation of Molecular Weight of Oligonucleotides 524
27.5 Amino Acids 525
27.6 Spectrophotometric Conversions 525
27.7 DNA Molar Conversions 525
27.8 Protein Molar Conversions 526
27.9 DNA-Protein Conversions 526
27.10 Molarity 526
27.11 Calculation of Mean
27.12 Variance and SD 529
References 530
Index 531
