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

Transgenic Mice (not nec. KO)

“Bacteria (KO)”

Transgenic Mice (KO) – view 2

Transgenic Mice (KO)

Phenocopying mutation by dsRNA injection Other psuedo-KO approaches…

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

Morpholino oligos Phenocopying mutation with...

RNAi in nematodes

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

Binary system – Gal4 UAS (expression mosaic)

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

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.)

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

Gal4- > Label or alter

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,

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

Enhancer trap principle

Random mutagenesis using a transposable element to create disruptions

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+

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

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

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

Just place embryos In X-Gal solution

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

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)

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

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

BRAINBOW

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

Zn Finger Nuclease

Compare ZNF and TALEN proteins on DNA at same scale

OUT- MCB 8.5 Transgenics

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

F3 screen for recessive mutation

F3 screen for recessive mutation

F3 screen for recessive mutation

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

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