Unit 2a Molecular Tools in Developmental Biology What we ll cover We ll only cover the minimum tools we need to study embryos We won t cover DNA cloning PCR or other common molecular biology techniques these are best left for another class We will study techniques that allow us to a assess when and where genes are expressed and b perturb gene expression or function The Central Dogma This means that we can study a protein by working with the DNA that encodes it DNA is far easier to work with than RNA or proteins Campbell 6e Fig 17 3 Nucleic Acid Hybridization see Purves et al 4e Fig 13 1 Nucleic Acid Hybridization Hybridization means that we can use sequences complementary to one we care about as probes It also allows us to design primers in procedures such as PCR see Purves et al 4e Fig 13 1 Genomic DNA vs RNA vs mRNA Genomic DNA regulatory and intronic sequences Enhancer distal control elements DNA Poly A signal sequence Proximal control elements Exon Upstream Intron Exon Promoter Primary RNA transcript 5 Intron Exon Downstream Transcrip on Exon Intron Exon Intron Exon RNA processing Intron RNA mRNA no intronic sequences Termina on region Cleaved 3 end of primary transcript Poly A signal Coding segment mRNA 5 Cap 5 UTR Start codon Campbell 8e Fig 18 8 Stop codon 3 3 UTR Poly A tail Making cDNA Genomic DNA is needed if we want to study a gene s regulation cDNA is useful is we just want to study the protein encoded by a gene cDNA no intronic sequences but easy to work with adapted from http 10e devbio com image php id 127 Make double stranded In Situ Hybridization Gilbert 8e Fig 4 16 In Situ Hybridization In situs are useful for determining where and when in an embryo a mRNA is expressed Gilbert 8e Fig 4 16 In Situ Hybridization Sea urchin gastrula stained for LvS1 mRNA J Hardin Indirect Immunostaining Immunostaining is useful for determining where and when in an embryo a protein is expressed From Becker et al Guide to Microscopy 5e Fig 13b Indirect Immunostaining J Hardin Red polyclonal antibody LvSnail Green monoclonal antibody 6a3 DNA Tagging a protein with GFP promoter Fusion protein DNA encoding protein of interest DNA DNA encoding encoding GFP 3 UTR transcription translation We add the DNA encoding GFP to the front Nterminal or back C terminal end of DNA encoding a protein we care about This encodes a fusion protein that is a combination of our protein and GFP that we can now see with a fluorescence microscope Confocal Microscope Setup Confocal microscopy is useful for removing out of focus fluorescence from thick specimens like embryos http www microscopyu com articles confocal confocalintrobasics html Overexpressing mRNA Overexpressing a normal or engineered protein can test sufficiency of a molecule for a developmental process Adapted from Kalthoff 1e Fig 4 18 Removing the function of a gene protein Taking away molecular function allows us to test necessity of a molecule for a developmental process Two methods act at the level of mRNA anti sense oligonucleotides and RNA mediated interference RNAi One method acts at the level of proteins morpholino antisense oligonucleotides morpholinos RNA Interference RNAi Double stranded RNA is introduced into a cell Double stranded RNA is chopped into pieces by an enzyme inside the cell in some animals like C elegans or we start with short interfering RNAs siRNAs Courtesy of Nature journals RNA Interference RNAi Guide mRNA AAAAA The pieces guide a set of proteins to the normal mRNA Mature mRNA RNA induced silencing complex RISC The normal mRNA is clipped into pieces and destroyed Courtesy of Nature journals Guide mRNA RNA Interference RNAi E cadherin WT E cad RNAi Gilbert 8e Fig 4 23 Morpholinos translation start site X 5 mRNA for Gene X 3 morpholino oligonucleotide against Gene X Chemically modified oligonucleotides bind to and prevent translation of a mRNA Note sometimes morpholinos block splicing of mRNAs Intentionally altering the genome genetic engineering We can add genetic material to make transgenics We can remove genetic material by targeted deletion of a gene to make a knockout In mice this can be accomplished via homologous recombination using engineered ES cells Starting with a known gene like this is called reverse genetics We can alter genetic material by inducing mutations and looking for desired phenotypes Starting with a phenotype and working back to the affected gene is called forward genetics Transgenic fish Reuters Transgenic Mice Kalthoff 2e Fig 15 21 Transgenic Mice Kalthoff 2e Fig 15 21 Transgenics carry extra DNA Transgenic mice Campbell 5e p 364 GFP mice Transgenic Cats Discovery Channel News Transgenic marmosets 2009 Kei and Kou Sasaki et al 2009 Nature 459 523 527 Making targeted deletions by homologous recombination We can make a knockout via homologous recombination using engineered ES cells in four steps a Make a piece of DNA targeting vector that can be inserted at the site of the normal gene via recombination b Use double drug selection to identify ES cells in which such exchange of DNA has occurred c Place these cells into a recipient blastocyst to make a chimeric embryo d Breed mice looking for mice in which engineered cells have made oocytes or sperm the knockout allele has made it into the germline Making knockout mice by homologous recombination Making the targeting vector a drug resistance gene is inserted into a normal copy of a gene Adapted from Capecchi 1994 Sci Am 270 3 52 9 Making knockout mice by homologous recombination If no insertion occurs then the cell can t survive the first drug Making knockout mice by homologous recombination If homologous recombination does NOT occur this gene is not clipped out then the cell IS sensitive to the second drug Making knockout mice by homologous recombination A second gene that confers sensitivity to another drug is added to the ends of the engineered gene Only if homologous recombination occurs will this gene be clipped out then the cell is NOT sensitive to the second drug Making knockout mice by homologous recombination Double drug selection for homologous recombination Making knockout mice by homologous recombination Inject engineered cells into a host blastocyst to make a chimera Making knockout mice by homologous recombination Breed to obtain homozygous knockouts in the germline Adapted from Capecchi 1994 Making knockout mice by homologous recombination Adapted from Capecchi 1994 int 2 knockout mouse an early example of a KO Making knockout mice