Chapter 3 Methods in Molecular Biology and Genetic Engineering

1. Chapter 3 Methods in Molecular Biology and Genetic Engineering

2. 3.9 Blotting Methods • Southern blotting involves the transfer of DNA from a gel to a membrane, followed by detection of specific sequences by hybridization with a labeled probe. • Northern blotting, RNA is run on a gel. • Western blotting entails separation of proteins on an SDS gel, transfer to a nitrocellulose membrane, and detection proteins of interest using antibodies.

3. FIGURE 21: Southern blot: Identifying Specific DNA Fragments (Edward Southern--the pioneer) Gel is soaked in alkali buffer to denature DNA or gentle vacuum pressure Drying or exposure to UV light Probes: Isotope or chemical

4. Northern blotting is similar to Southern blotting, but involves the transfer of RNA from a gel to a membrane RNA

5. How to separate mRNA from all other classes of RNA

6. How to separate mRNA from all other classes of RNA mRNA contains ~200 oligo(A) residues at 3’ end FIGURE 22: Poly(A)+ RNA can be separated from other RNAs by fractionation on an oligo(dT) column

7. Northern blotting: Measuring gene activity Poly(A)+ RNA: from rat tissues Probe: G3PDH (glyceraldehyde-3-phosphate dehydrogenase) From Dr. Yu

8. Western blotting • Western blotting entails separation of proteins on an SDS gel, transfer to a nitrocellulose membrane, and detection proteins of interest using antibodies. wikipedia

9. FIGURE 23: Western blot

10. 3.9 Blotting Methods • Antibodies can recognize the protein of interest or an epitope tag. • epitope tag – A short peptide sequence that encodes a recognition site (“epitope”) for an antibody, typically fused to a protein of interest for detection or purification by the antibody. Human influenza hemagglutinin (HA): YPYDVPDYA The HA tag is derived from the HA-molecule corresponding to amino acids 98-106 has been extensively used as a general epitope tag in expression vectors.

11. 3.10 DNA Microarrays • Gene expression array are used to detect the level of all the expressed genes in an experimental sample. • SNP arrays permit genome-wide genotyping of single nucleotide polymorphisms. =>use allele-specific oligonucledtide probe • Array comparative genome hybridization (array-CGH) allows the detection of copy number changes in any DNA sequence compared between two samples.

12. 3.10 DNA Microarrays • DNA microarrays comprise known DNA sequences spotted or synthesized on a small chip. FIGURE 24: Microarrays show the levels of all the expressed genes in an experimental sample.

13. 3.11 Chromatin Immunoprecipitation(ChIP) Sonication to 200~1000bp Chromatin immunoprecipitation (ChIP) allows detection of specific protein–DNA interactions in vivo. PCR or Blotting method

14. 3.12 Gene Knockouts and Transgenics • transgenics – Organisms created by introducing DNA prepared in test tubes into the germline. • The DNA may be inserted into the genome or exist in an extrachromosomal structure. FIGURE 26: Transfected DNA can be incorporated into the mouse genome Photo reproduced from P. Chambon, Sci. Am. 244 (1981): 60-71. Used with permission of Pierre Chambon, Institute of Genetics and Molecular and Cellular Biology, College of France.

15. 3.12 Gene Knockouts and Transgenics • ES (embryonic stem) cells that are injected into a mouse blastocyst generate descendant cells that become part of a chimeric adult mouse. • When the ES cells contribute to the germline, the next generation of mice may be derived from the ES cell. • Genes can be added to the mouse germline by transfecting them into ES cells before the cells are added to the blastocyst.

16. Defective genes can be replaced by functional genes using transgenic techniques GnRH (gonadotropin-releasing hormone) GAP (GnRH-associated peptide)

17. The Nobel Prize in Medicine 2007 University of Utah, Salt Lake City, UT, USA, Howard Hughes Medical Institute Cardiff University, Cardiff, United Kingdom University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Forthediscoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells www.nobelprize.org

18. 3.12 Gene Knockouts and Transgenics FIGURE 28: ES cells can be used to generate mice

19. 3.12 Gene Knockouts and Transgenics • An endogenous gene can be replaced by a transfected gene using homologous recombination. • The occurrence of successful homologous recombination can be detected by using two selectable markers, one of which is incorporated with the integrated gene, the other of which is lost when recombination occurs.

20. TK: Thymidine Kinase (sensitive to gancyclovir) TK phosphorylates gancyclovir, which make it toxic Homologus recombination involves two exchanges within the sequence of donor gene FIGURE 29: Transgenes can be selected in ES cell

21. 3.12 Gene Knockouts and Transgenics Gene Knockout: Gene deletions Gene knock-in: Replacement of a gene with alternative form Gene Knockdown: Reduce the amount of gene product The Cre/lox system is widely used to make inducible knockouts and knock-ins.

22. Gene Knockout FIGURE 30: Cre excises the sequence between lox sites Structure from Protein Data Bank: 1OUQ. E. Ennifar, et al., Nucleic Acids Res. 31 (2003): 5449-5460.

23. FIGURE 31: Cre/lox excises a target only when Cre is activated

24. Cre/lox system

25. Cre/lox system 34-base loxP sequence cre.jax.org

26. Gene Knock-in FIGURE 32: A knock-in replaces an endogenous gene with an alternative sequence

27. knock-in involves a gene inserted into a specific locus, and is a "targeted" insertion

28. Inducible Gene Expression and Gene Modification in Transgenic Mice Tetracycline-Inducible Systems JAISSER F 2000

29. Inducible Gene Expression and Gene Modification in Transgenic Mice Cre/lox system and its use as an inducible expression system Cre-ER (ER:Estrogenreceptor)system JAISSER F 2000

30. Brainbow

31. Brainbow Livet J et al., 2007

32. Brainbow Livet J et al., 2007

33. Brainbow Livet J et al., 2007

34. Brainbow Livet J et al., 2007