Biotechnology Techniques in Developmental Biology Ch. 5 - Gilbert pp.86-98

Today’s Objectives Identify the ways that modern Molecular Biology/Biotechnology techniques have greatly advanced Developmental Bio. Research Relate the Recombinant DNA techniques learned at BTHS for the past 3 years to the field of Dev. Bio.

Huge Step Forward in Developmental Bio Research The advent of Recombinant DNA technology! –Name some common techniques... Before this, embryologists were limited to cutting and pasting, tracking cells, culturing in a dish, etc. (see Historical Perspectives slides)

Why is the use of Rec. DNA Technology so useful? Major question for embryologists revolved around Genomic Equivalence... If every nucleus in every cell in the embryo contains the SAME genetic info, then HOW do we get cells that are differentiated into specialized cell types? WE NOW KNOW ->

Recombinant DNA and Biotechology Techniques Allow us to: –Find genes that are expressed in the embryonic cells –Analyze the expression of genes (&proteins) in different cells in the embryo! –Analyze FUNCTIONS of genes (& proteins)

Cloning a gene! Recombinant DNA technology basics –Finding the DNA sequence of a gene of interest and splicing it into a plasmid –Making millions of copies of that gene (the actual DNA), so we can further work with it

Determining Functions of a Gene in an Embyro Think of it like gathering evidence at a crime scene - we need a few clues! –1) Where is the gene expressed? –2) What happens if we give a cell that doesn’t normally express the gene, the ability to express it? (OVEREXPRESSION) –3) What happens if we take a normal cell and remove the gene? (KNOCKOUT)

Finding out WHERE a gene is being expressed: Can’t look at the DNA for this: 1) Find the mRNA that has been transcribed from the DNA 2) Find the protein that has been translated from the mRNA If the gene is EXPRESSED in a cell/tissue/organ, it probably has a FUNCTION there

Finding mRNA that is expressed in a cell/embryo 1) Northern blotting: –Isolate total mRNA, run a gel, blot to nitrocellulose filter, probe for RNA you are interested in with the complementary sequence (hybridization) Must dissect out the tissue/cells that you are interested in and isolate RNA from those cells only –Ex. - heart tissue, ectoderm, etc.

Finding mRNA that is expressed in a cell/embryo 2) RT-PCR –RT stands for Reverse Transcriptase –What does PCR usually enable us to do? –RT-PCR enables us to: (p. 87)

Can analyze miniscule amounts of RNA from a tissue

Finding mRNA that is expressed in a cell/embryo RT-PCR only allows us to look at 1 gene at a time. 3) Microarray technology (commonly known as __________) Enables researchers to examine thousands of mRNA transcripts simultaneously - Very Powerful! –Ex. Could look at a normal embryo and a mutant embryo and compare EXACTLY which genes are turned on and off

Finding mRNA that is expressed in a cell/embryo In all of these cases, you are working with mRNA that was EXTRACTED out of cells or tissues - can’t see it IN the embryo 4) In situ hybridization: enables researcher to visualize expression of a gene INSIDE of a whole embryo (not alive)

In situ Hybridization (p ) Works just like a Northern blot, but probe soaks into the embryo, then an antibody attaches, and an enzyme turns the embryo purple wherever there is mRNA expressed!

Finding a Protein expressed in embryo Western Blotting –Grind up embryonic tissue –Run through SDS-PAGE –Blot onto nitrocellulose –Incubate with primary antibody specific for a protein –Then secondary antibody to primary antibody enabling colorimetric enzyme reaction

Immunohistochemistry Works just like a western, but antibodies are applied to the ENTIRE embryo (or slides containing sections of an embryo) Binds and identifies the areas where that specific protein is being expressed! –Very powerful tool for embryology

Manipulation of Genes INSIDE of embryos! Transgenic cells and organisms can be created We better understand the function of a gene by FORCING a cell to express that gene, and looking for phenotypic changes We can gain even greater insight by REMOVING the function of a normal gene, and examining the phenotypic outcome

Transgenic Organisms Inserting foreign genes into organisms –Microinjection –Transfection –Electroporation –Transposable elements –Retroviruses Can help to study gene function

Production of Transgenic Mice

Chimeras Creating embryos that are mixtures of cells –Quail-Chick chimeras (LeDouarin) –Mouse chimeras - early, using embryonic stem cells Blastula stages Can deliver transgenic genes Pass transgene to germ line Get next generation to express gene in ALL cells

Gene Targeting in Mice Step beyond Transgenic mice Use homologous recombination to replace a normal gene with a non- functional copy Knock out function of that gene - don’t make functional protein Extremely valuable technique for finding out about gene function!

Technique for Gene Targeting

Morphological Analysis of Bmp7 Knockout Mice

Disadvantages of KO mice Takes a long time! Expensive! May not get a phenotype –Genetic redundancy - multi-gene families

More ways to Silence Gene Function Antisense RNA (single stranded) –Incubate with complementary RNA strand –Binds to mRNA that is expressed and keeps it from being translated –Temporary (instable) Morpholinos –Similar to antisense RNA –Antisense Oligomers - small pieces of DNA or RNA –More stable than antisense RNA RNAi

Discovered by Andrew Fire and Craig Mello in C. elegans- recently awarded Nobel Prize! Insert double stranded RNA into cells/organisms Results in inhibition of translation of mRNA - no functional protein gets made Functional Knockouts

Injection of dsRNA for E-Cadherin into the Mouse Zygote Blocks E-Cadherin Expression

Determining Functions of a Gene in an Embyro Think of it like gathering evidence at a crime scene - we need a few clues! –1) Where is the gene expressed? –2) What happens if we give a cell that doesn’t normally express the gene, the ability to express it? (OVEREXPRESSION) –3) What happens if we take a normal cell and remove the gene? (KNOCKOUT)