Animal Development Lecture Notes PDF

January 9 th Dev Biol Lecture Intro to Model Organisms Use of Genetics Common Techniques used  Model Organisms Species Common name C. elegans Worm Drosopholia m. Fruit Fly Brachydanio rerio Zebrafish Xenopus laevis African clawed frog Gallu...

January 9 th Dev Biol Lecture Intro to Model Organisms Use of Genetics Common Techniques used  Model Organisms Species Common name C. elegans Worm Drosopholia m. Fruit Fly Brachydanio rerio Zebrafish Xenopus laevis African clawed frog Gallus domestica Chicken Mus musculus Mouse  Model Organisms Blastopore Involution (blastopore) forms at anus forms at mouth  easy to obtain n >1000 eggs for first four species Cost C. elegans = Chick < Drosophila = Zebrafish = Xenopus < Mouse  Access to embryos Xenopus and zebrafish best (external fertilization, embryos free-living) C. elegans is good Chicks, egg has # of cells when laid Developmental Milestones  Forward genetics Defining mutation: -determine the gene of interest by positional mapping it onto chromosome -clone the g. of i. -insert clone into mutant: Rescue?  Genetic pathways Mutations in a,b or c cause phenotype Embryo with b mutation gene a does not rescue replacement of gene b or expression of gene c (more than normal) allows for formation of Segment 3 Repression as a way of Activation a b c  Repression as a way of Activation c (forms segment) Repression as a way of Activation b c ( does not form segment) Repression as a way of Activation a b c (forms segment) Two negatives equal a positive ! Activation usually repression of a repressor  Maternal vs Zygotic Determinant WT mother makes determinant and gives to egg Mutant mother can't make determinant (but itself is okay !) Thus, offspring Sperm (-,-) is affected (1 generation removed) Maternal effect mutation  Techniques Dissecting Compound  Fluorescent microscopy fluorochrome absorbs light of a particular energy and emits light of a lower energy e.g. GFP, RFP, Cy3 and Cy5  Study of gene expression Need to know normal expression patterns of genes - stage of development - region of embryo - quantification Biochemical methods both mRNA and protein  Northern blot or SDS-PAGE/western stage series for specific gene product (either mRNA or protein) In situ hybridization: for detection of mRNA (AP = alkaline phosphatase) Immunostaining: for detection of protein  Wholemount Chick Embryo stained for Pax6 Wholemount Section  Reporter Genes GFP fuse reporter to promoter of genes: expressed in particular cell types LacZ  Regulatory regions of the mouse Pax6 gene promoter consists of modular regions (function independently) LacZ Expression: pancreas, lens & cornea, and neural tube above plus LacZ retina  Microarrays: -examination of large numbers of gene products at once -compare 2 different samples which down genes are upregulated duetomutants Array can contain partial DNA (oligos) of entire genome wildtypeHybridization of cDNA to express Array both no change in gene apron ofwildtype mutout mutant  exp Quantify intensity of gene expression upon "treatment"  targetin RNAseq modern thing wholespecific the - whole transcriptome shotgun sequencing sequence every mRNA - presence and quantities of RNA in sample ChIP-Seq Mardis, E.R. Nat. Methods 4, 613-614 (2007) ChIP-Seq where the TEs directly affect the gene  knock out CRISPR-Cas9 a type of Novel way to generate gene deletions Allows for creation (or repair!) of mutations make direct mutants Ex reverse genetics Care cuts dsDNA gRNA guide RNA has the gene that you want to cut with the Cas9