Developmental Genetics Lecture PDF

Developmental genetics extensive use of mutants mutations can be: spontaneous induced (chemical, radiation) targeted  alleles: different versions of a gene one normal gene = wild-type Total nuclear DNA = genome specific combination of alleles = genotype Total...

Developmental genetics extensive use of mutants mutations can be: spontaneous induced (chemical, radiation) targeted  alleles: different versions of a gene one normal gene = wild-type Total nuclear DNA = genome specific combination of alleles = genotype Total characteristics of an organism = phenotype Allelic series  Forward genetics phenotype find gene with mutation Reverse genetics create mutation define in specific gene phenotype  Loss of function = protein less active than wild-type Gain of function = protein constitutively active  Determining the Function of Genes during Development Transgenic cells/organisms... express (overexpress) gene of interest or knock it out microinjection transfection electroporation retroviruses RNA interference Production of transgenic mice Technique for gene targeting Production of transgenic mice  Heterozygous transgenic mice +/+ (25%) +/- (50%) -/- (25%)  Analysis of Bmp7 knockout mice BMP7 = Morphogen, function depends on context  knockout also for addRNA interference + dsRNA to cells (seq specific) to thegene youwant Complementary to knock out dsRNA is cleaved to small fragments (siRNA) siRNA binds target mRNA mRNA degraded or Micro RNA in genone prevented from siRNA artifical to translating way research gene expression Cell-Cell Signaling in Development Control of differential expression receptor binds activated induce Ligands receptors transducing signals modified protein or 2ⁿᵈ messenger nucleus or cytoskeletonFenter  Mechanisms of Inductive interaction Contact-Dependent Paracrine diffusible Juxtacrine inductions  Classes of Receptors Ligand small and lipid soluble Tehreane receptor becomesa transcription factor diffusethrough nuclearmembrane receptoruntil Nuclear Receptor LigandsExamples steroids T E PR retinoicacid  Classes of Receptors Tyrosine kinases or Ser/Thr kinases Tusually directlylinkedtoreceptor Taddphosphates to OHgroups Enzyme-linked receptor hencephosphorylation Ligand binding domain (exterior) Single transmembrane region Enzyme active site  Classes of Receptors or or or G proteins trimeric G-protein-linked receptors α β I subunite highest eceptor in the body or momeric Classes of Receptors  TGF-b superfamily Tigand or TGFb pathway Note Type I 2R are kinases that phosphorylate on either the Serine Threanine of Smad  Belgian Blue myostatin TGFB lingand  Tyrosine Kinase Family receptor kinase Tyrosine smell c 0 1 FGF - Fibroblast Growth Factor Eph Receptor Ex Mckinase pathway activatetranscriptionfactor  Hedgehog pathway Patched Smoothened No hh t pbc Lts a smo t Gli a generegulation hh Lh bindsptc c ptc t Smo Ils Gli c generegulation  Cyclopic lamb sonic hedgehog Shh importantfor developing midlineof animal no cholesterol no Shh wings no you done in mice Wingless of Wnt Family Frizzled at 93 b-catenin Dishevelled inactivatedby gsk t gene expression Notch/Delta Family Genetic Mutations Notes created on January 22, 2025 at 12:21 PM by Minutes AI Gene Function and Phenotype Association (00:00 - 09:59) Forward genetics Develop a phenotype to find the gene mutation. Induce random mutations by offending or spontaneous methods. Find a phenotype of interest and work back to identify the gene mutation. Reverse genetics More targeted approach to study the effect of a specific gene. Create an application in a specific gene to define the phenotype. Types of Genetic Mutations Loss of function mutations Result in a protein that is less active or completely inactive (null). Often recessive; heterozygous individuals may not show phenotype due to wild Gain of function mutations Can turn a protein on constitutively, leading to overactivity. Often dominant; phenotype may be observable in heterozygous individuals. Transgenic Cells and Organisms Definition of transgenic Adding a gene to either cells or organisms. Methods of creating transgenic organisms Micro injection: Directly inject plasmid into individual cells. These notes were taken with Minutes AI (https://myminutes.ai)  Transfection: Use of chemicals to facilitate DNA incorporation into cells. Electroporation: Applying current to cells to create pores for DNA entry. Example: Creating a Transgenic Mouse Goal: Knock out a protein, specifically BMP7. BMP7 stands for bone morphogenic protein number seven. Importance of BMP7 Further details will be discussed during the course. Embryonic Stem Cells and Plasmid Construction (09:59 - Started with a pregnant mouse and an early embryo known as a blastocyst. The blastocyst contains a layer of cells and an inner cell mass, which consists of Culturing embryonic stem cells can be done indefinitely. A plasmid is created that includes the BMP7 gene. The NEO R gene, which stands for neomycin, is inserted into the BMP7 gene. Neomycin Resistance Neomycin is an antibiotic that can kill untreated cells at high concentrations. The NEO R gene encodes an enzyme that breaks down neomycin. Cells that take up the plasmid survive in the presence of neomycin. This process serves as a selection marker for modified cells. Homologous Recombination The plasmid must be incorporated into the genome to persist in mammalian cells. This incorporation occurs through homologous recombination. The efficiency of this process is low, potentially one in 50 to one in 100 cells. These notes were taken with Minutes AI (https://myminutes.ai)  Neomycin is added to select for cells that have undergone successful modification. Verification and Mating Process Genotyping is performed to confirm successful incorporation of the plasmid. PCR is used to ensure the sequence is as intended. Modified embryonic stem cells are injected into a second blastocyst. The blastocyst is then implanted into a pseudopregnant mouse. A vasectomized male is used to mate with the female mouse to prepare the Blastocyst Transfer and Implantation (20:06 - 30:04) Transfer of blastocyst into the uterus relies on luck for implantation. Approximately one in three blastocysts implant successfully. Successful implantation can lead to chimeric mice. Chimeric mice consist of cells from both mutated and normal embryonic stem Germline Formation in Chimeric Animals Need to hope that mutated cells form the germ line. Germ line mutations occur in sperm or eggs about 10-20% of the time. Testing for germline mutations requires crossing chimeric animals with wild type. If germline is mutated, it will produce heterozygous transgenic mice. Mendelian Genetics in Offspring Crossing heterozygous mice should show Mendelian ratios. Expected ratios: 25% homozygous wild type, 25% homozygous mutant, 50% Phenotypes and Gene Function These notes were taken with Minutes AI (https://myminutes.ai)  Phenotypes can indicate the type of mutation. Gain of function mutations may present phenotypes, while loss of function often Essential genes may lead to lethality if knocked out. Analysis of offspring can reveal genotypic distributions before birth. BMP7 Mice Analysis BMP7 is crucial for eye and kidney development. Knockout mice show no eyes and total kidney atrophy. Caution is advised regarding gene names and their functions. Names may not accurately reflect the protein's role in development. Phenotypes and Gene Knockout (30:05 - 39:54) Importance of observing phenotypes in gene knockout studies. Example: Knocking out BMP7 may not show a phenotype. Possible reasons for not observing a phenotype. "The protein couldn't be translated correctly." Redundancy in the genome: multiple proteins may perform the same function. Example: BMP6 may compensate for the absence of BMP7. Background mutations could affect gene expression. Gene Functions and CRISPR Genes may have multiple roles. Some genes are referred to as "pleiotropic," performing multiple functions. Introduction of CRISPR technology. CRISPR is a more efficient method for gene knockout in embryonic stem cells. Increases likelihood of obtaining embryonic stem cells with desired mutations. These notes were taken with Minutes AI (https://myminutes.ai) RNA Interference Overview of RNA interference as a gene knockout method. Involves adding double-stranded RNA complementary to the target mRNA. Mechanism of action. Double-stranded RNA is cleaved into small interfering RNAs (siRNAs). siRNAs can degrade mRNA or inhibit its translation. Differences Between siRNAs and miRNAs Distinction between small interfering RNAs (siRNAs) and microRNAs (miRNAs). siRNAs are exogenous, introduced by researchers. miRNAs are endogenous, naturally occurring within the genome. Functionality. siRNAs are used experimentally to manipulate gene expression. miRNAs regulate gene expression as part of normal cellular processes. Signal Transduction Introduction to signal transduction and cell signaling. Important for understanding cell interactions during development. Basic mechanism of signal transduction. A ligand produced by one cell binds to a receptor on another cell, activating the Cell Signaling Mechanisms (39:55 - 49:52) Ligands can induce or transduce signals to other proteins within the cell. These signals modify protective proteins or create second messengers. Resulting modified proteins or second messengers can activate gene Cell interactions can be classified as: These notes were taken with Minutes AI (https://myminutes.ai)  **Contact-dependent**: Ligand remains attached to the first cell and interacts **Paracrine**: Ligand is secreted into the extracellular matrix and diffuses to bind Types of Receptors Receptors can be classified based on their interaction with ligands. **Nuclear Receptors**: Ligands are membrane soluble and can pass through the These receptors form a ligand-receptor complex that can affect gene Example ligands include hormones like testosterone and estrogen. **Enzyme-linked Receptors**: Ligand binds to the receptor linked to an enzyme, often Kinases add phosphate groups to proteins, activating signaling pathways. Example: Fibroblast growth factor receptor. G Protein-linked Receptors G protein-linked receptors are the most common type in the body. They play a significant role in development and daily physiological functions. Named for their association with guanine nucleotide binding (GDP or GTP). They are trimeric proteins, with different types such as G, I, and Q. Second Messenger Systems (49:52 - 59:41) Increase in cyclic AMP Activates receptors leading to various cellular processes. Increase in calcium concentration Usually sourced from the endoplasmic reticulum. Receptor Types Ligand binding receptors These notes were taken with Minutes AI (https://myminutes.ai)  Create ion channels that allow ions like calcium, sodium, chloride, and potassium TGF Beta Superfamily Involved in development and cancer. TGF beta is the canonical ligand. Receptor types TGF beta binds to type 1 and type 2 kinase receptors. Phosphorylates SMAD proteins, which are transcription factors. Myostatin mutation Leads to double muscle phenotype in Belgian Blue bulls. Mutation causes excessive muscle development but results in health issues. Tyrosine Kinase Family Fibroblast growth factor (FGF) as a ligand. Activates tyrosine kinase receptors. Activation of RAS protein RAS is a small G protein, mutated in many cancers. Activates the MAP kinase pathway, influencing gene expression. Hedgehog Pathway (59:42 - 1:07:40) Initially found in Drosophila, named "Hedgehog" due to mutations causing pointy The receptor is called "patched," which inhibits "smoothened." Smoothened interacts with the transcription factor "Gly." Hedgehog inhibits patched from inhibiting smoothened, leading to activation of Gly. Sonic Hedgehog These notes were taken with Minutes AI (https://myminutes.ai)  In vertebrates, there are three Hedgehog ligands: Sonic Hedgehog, Desert Hedgehog, Sonic Hedgehog is the most well-known, named after Nintendo's character. Cyclopic phenotype observed in a lamb due to maternal consumption of a plant Cholesterol is important for Sonic Hedgehog function and development. WNT Pathway Discovered in Drosophila, named from "wingless" and "int" from mice. WNT ligand binds to the receptor "brizzled." Activates beta-catenin, which is normally phosphorylated by GSK3 to remain WNT activation inhibits GSK3, allowing beta-catenin to activate gene expression in the These notes were taken with Minutes AI (https://myminutes.ai)