2 - Genetics & Sexual Reproduction

reverse genetics

a specific gene is altered and the phenotype is investigated

genotype —> phenotype

forward genetics

examination of the genetic cause of an altered or abnormal phenotype

phenotype —> genotype

forward genetic screens

ENU mutagenesis randomly causes DNA damage so one can select for phenotypes of interest and go back to sequence the genome to find the mutation

RNA interference

• A double stranded RNA (DICER) —> single stranded small interfering RNAs (siRNAs) —> hybridize against the target gene’s mRNA —> degradation

• fast & easy way to study gene function through targeted mutagenesis

• problem: off target effects, temporary gene knockdown

genetic screen

examining thousands of mutagenized ppl to find a phenotype of interest (organisms must reproduce rapidly)

how to screen for mutations that cause lethality

• temp sensitive mutants

• colonies replicated onto two identical plates & incubates at two diff temps

• mutant colony in which cells proliferate at the cooler temp but fail to at the warmer temp

complementation test

• tests whether a mutation is in two diff genes or one

• when an albino bird is bred with another from different strains, the resulting offspring have normal coloration

• means that albinism results from recessive mutations in diff genes

polymorphisms

sequence variations at specific regions of the genome

• most are single nucleotide polymorphisms (SNPs)

• other are copy number variations (CNVs)

• inherited on haplotype blocks (large segments of DNA)

how do haplotype blocks trace ancestry?

• more distantly related ppl will have diff haplotype blocks due to recombination that occurs over time

• close relatives share haplotype blocks bc less cross over events occur

mendelian disorders

caused by mutations in a single gene

dominant mendelian disorder

mutation in 1 allele enough to cause disease

recessive mendelian disorder

mutation in both alleles needed to cause disease

Genome wide association studies (GWAS)

identify DNA variations that are more frequent in ppl with age related degeneration

How to model the mutation’s effects to determine if it is disease causing or not?

• identify impact of mutation on global gene expression

• RNA sequencing

  • RNA —> cDNA + sequenced

  • quantitative analysis of cell’s transcriptome

  • detects rare splice variants

in situ hybridization

tells when and where a gene is expressed by hybridizing a fluorescent single stranded probe against complementary RNA sequences

reporter genes

• used to determine pattern of a gene’s expression

• coding sequence of gene replaced with a reporter gene (ex. GFP)

• GFP controlled by gene’s regulatory sequences to ensure that its expression matches the normal expression patterns of the target gene

homologous recombination w/ embryonic stem cells

• targeted gene replacement or gene knockout

• gene sequence altered in cultured ES cells

• DNA plasmids introduced into these cells w/ a mutated piece of DNA flanked by homologous sequence for genome

• antibiotic resistance gene selection marker also added

• ES cells cultured in presence of antibiotic

• pick surviving cells and inject into blastocyst

• result in chimeras

CRISPR

• bacterial enzyme Cas9 which induces double stranded breaks in DNA

• guide RNA needed to target specific sequences

• donor DNA added w/ altered DNA as a template for homologous repair

• homologous recombination swaps the wild type gene for donor gene & repair enzymes

conditional knockouts (CKO)

• gene selectively disabled in a target tissue

• mouse 1: insert 2 LoxP sites into flanking regions of target gene

• mouse 2: insert DNA encoding for Cre recombinase downstream of gene promoter

• mate mouse 1 & 2: offspring that inherit gene flanked by sites and cre-recombinase have excision of gene

Cas9 can also be used..

to activate a normally dormant gene or turn off an actively expressed gene by using a mutant form that no longer cleaves DNA