1. Transgenic Mice (not nec. KO) 1. How to get the DNA in? 2. Where is it, once inside?

2. Transgenic Mice (not nec. KO)

4. “Bacteria (KO)”

5. Transgenic Mice (KO) – view 2

7. Transgenic Mice (KO)

11. Phenocopying mutation by dsRNA injection Other psuedo-KO approaches…

12. Phenocopying mutation by expressing... Other psuedo-KO approaches… Double stranded RNA (RNAi) hairpin two complementary RNAs Antisense Dominant negative Encoded neutralizing antibody

13. Morpholino oligos Phenocopying mutation with...

14. RNAi in nematodes

16. Binary Systems Cre LoxP - genetic changes Gal4 > UAS - pseudo-genetic (transcriptional activation)

17. Binary system – Gal4 UAS (expression mosaic)

18. Binary system – Cre Lox (mosaic - somatic genetic alteration)

19. Binary system – Gal4 UAS (expression mosaic) Or: pseudeo-KO with UAS RNAi or DN, or... Or: Neuronal mapping and connectomics, with- UAS- nls-GFP (soma); UAS-cd4 (mb.)

20. VT line enhancer ‘bashing’ Gal4 driven sequence for many separate, discrete enhancer elements per gene

21. Gal4- > Label or alter

22. Figure 1 Binary Systems (A) Overview of a binary system. The transactivator binds to a binding site to activate a responder. Expression of a repressor blocks the activity of the transactivator. Compounds (C) can modulate the activity of repressor and tran... Koen J.T. Venken, Julie H. Simpson, Hugo J. Bellen Genetic Manipulation of Genes and Cells in the Nervous System of the Fruit Fly Neuron, Volume 72, Issue 2, 2011, 202 - 230 http://dx.doi.org/10.1016/j.neuron.2011.09.021

23. Traps / Reporters: Generate huge numbers of Drivers Enhancer traps (reporters) Gene traps / promoter traps

24. Enhancer trap principle

25. Random mutagenesis using a transposable element to create disruptions

26. P: X P-element-{w+}, & transposon gene ; II ; III X ; II ; III Y F1:X ; II ; III Y w+ MALES Random mutagenesis: using a genetic element to create disruptions:an example with a transposon in fly X w+

27. P-element-mediated enhancer detection: a versatile method to study development in Drosophila. Bellen, HJ et al 1989

28. P-element-mediated enhancer detection: an efficient method for isolating and characterizing developmentally regulated genes in Drosophila Wilson,, C et al 1989

29. P-element-mediated enhancer detection: a versatile method to study development in Drosophila. Bellen, HJ et al 1989

30. Just place embryos In X-Gal solution

31. Enhancer trap lines In 1989 (Bellen), done with random enhancers directing {‘trapping’} lacZ (blue) Now, done with random enhancers directing {‘trapping’} Gal4 (driver) In mice, limited-replication retrovirus Enhancer traps

32. Combining ‘Traps’ and Binary systems Generate drivers (Cre, Gal4, etc.) through enhancer traps: just use driver instead of reporter Have the ability to drive ANY gene with a large collection of drivers (usually tissue specific)

33. Cloning genes from gene disruptions with genetic elements (transposon, retrovirus... ) First approaches: genomic libraries then “plasmid rescue” NOW – Inverse PCR

34. P-element-mediated enhancer detection: an efficient method for isolating and characterizing developmentally regulated genes in Drosophila Wilson,, C et al 1989

36. BRAINBOW

39. Directed mutagenesis NEW WAVE (last 5 years): Zn finger nucleases TALEN CRISPR/ Cas9

41. Zn Finger Nuclease

49. Compare ZNF and TALEN proteins on DNA at same scale

54. OUT- MCB 8.5 Transgenics

55. Generalized: “Forward” Genetics First - mutant/phenotype of significance Second – unravel molecular facts, basis

56. F3 screen for recessive mutation

57. F3 screen for recessive mutation

58. F3 screen for recessive mutation

59. F3 screen for recessive mutation: structures to screen in zebrafish for phenotypes

60. F3 screen for recessive mutation : And beyond… look for maternal- effect genes