1. 基因工程學 產物 : 重組蛋白質 蛋白質 DNA( 基因 ) mRNA 胰島素缺乏糖尿病 如胰島素, 干擾素等 (93 億美元, 北美,1997)

3. PCR: primer design ATTGCCACGTTGACGGTCAG CACGATTTCCGGAAGTAC

4. Inducer: IPTG, lactose

5. Restriction endonucleases Palindromes Blunt ends v.s. sticky ends

7. 重組蛋白質 在寄主 (host) 細胞中的表達 E. coli 染色體 質體 基因 優點 產量大 操作易 缺點 no post-translation modification inclusion body not secrete protein 4,000kb

8. 重組蛋白質 在寄主細胞中的表達 E. coligram-positive bacteria 缺點 B. subtilis not secrete proteinproteases limited vector & promoter “signal” has not found yet

9. 質體不穩定

10. Growth inhibition

12. 基因工程學 昆蟲細胞 染色體 基因 細胞核 病毒 基因治療 基因食物 receptor

13. 常見的動物細胞寄主 insect cell-baculovirus mammal-retrovirus chinese hamster ovary (CHO) cells immortal cell

14. Baculovirus Replication and Gene Expression Infection Gene Expression and Protein Synthesis: Immediately-Early Genes Delay-Early Genes Late Genes Very Late Genes: polyhedrin and p10 Virus Assembly and Release

15. Cotransfection with AcMNPV DNA Plaque Assay for putative recombinant virus Confirm presence of target gene by PCR or Southern blot Amplify and titer the recombinant virus Infect cultured insect cells

16. Generation of Recombinant Baculovirus by Bacmid

19. Molecular tools Gel Electrophoresis Labeled Tracers Hybridization Mapping and quantifying transcripts Measuring transcription rates in vivo Assaying DNA-protein interactions Knockouts

21. Pulse-field gel electrophoresis (0.2-2.2 Mb)

22. Two-dimensional gel electrophoresis

23. Ion exchange chromatography R + A - + B - R + B - + A -

24. Gel filtration chromatography

25. Affinity chromatography

26. Molecular tools Gel Electrophoresis Labeled Tracers Hybridization Mapping and quantifying transcripts Measuring transcription rates in vivo Assaying DNA-protein interactions Knockouts

27. Molecular tools Gel Electrophoresis Labeled Tracers Hybridization Mapping and quantifying transcripts Measuring transcription rates in vivo Assaying DNA-protein interactions Knockouts

29. DNA fingerprinting

30. Figure 5.18 The Sanger dideoxy method of DNA sequencing

32. Molecular tools Gel Electrophoresis Labeled Tracers Hybridization Mapping and quantifying transcripts Measuring transcription rates in vivo Assaying DNA-protein interactions Knockouts

33. S1 mapping the 5’-end of a transcript

34. Primer extension

35. Molecular tools Gel Electrophoresis Labeled Tracers Hybridization Mapping and quantifying transcripts Measuring transcription rates in vivo Assaying DNA-protein interactions Knockouts

37. Molecular tools Gel Electrophoresis Labeled Tracers Hybridization Mapping and quantifying transcripts Measuring transcription rates in vivo Assaying DNA-protein interactions Knockouts

39. Figure 5.35

40. DNase footprinting Page 125

41. Molecular tools Gel Electrophoresis Labeled Tracers Hybridization Mapping and quantifying transcripts Measuring transcription rates in vivo Assaying DNA-protein interactions Knockouts