2.2 - genetics + gene function

reverse genetics

1. select gene to investigate

2. mutate that gene using chem/molecular bio

3. observe phenotype w/ mutated gene

4. compare mutant + wild-type; gene of interest is likely responsible for phenotype changes

forward genetics

1. gather/create mutant individuals that display the phenotype of interest

2. screen the genomes of the mutant individuals + look for a gene mutation that all of the individuals share

3. if all have a mutation in the same gene, this gene is likely responsible for the phenotype

RNAi process

1. dsRNA processed by DICER into siRNAs

2. RISC complex recruits siRNAs

3. siRNA hybridizes against target gene’s mRNA

4. mRNA degraded

RNAi characteristics

fast + easy way to study gene function through targeted mutagenesis

• problem = off target effects, only temporary gene knockdown

conditional mutants

created by scientists that express a mutation under specific conditions

• popular method = insert mutation downstream of a promoter which is controlled by transcription factors relevant to the condition

• permit study of lethal mutations

polymorphisms

sequence variations at particular regions of the genome

• most are SNPs, others include CNVs

• tend to be inherited on haplotype blocks

polymorphisms and family history

• distant relatives = diff haplotype blocks from meiotic recombination

• close relatives = share numerous haplotype blocks

mendelian disorders

caused by mutations in a single gene + therefore the mutation segregates w/in families in a mendelian fashsion

dominant disorders

mutations in 1 allele is sufficient to cause the disease

recessive disorders

mutation in both alleles is required to cause disease

• more prevelent in certain parts of the world bc of incest

multigenic disease

usually arise from cumulative effects of mutations in multiple genes that normally have a small effect size, but together create a large enough effect

• tend to arise later in life

• risk-enhancing alleles are inherited rather than eliminated; can become common

how are risk alleles identified

by looking for Single Nucleotide Polymorphisms that are statistically linked + more common to the development of the disease

genome-wide association studies (GWAS)

identify DNA variations that are significantly more frequent in ppl w/ age-related macular degeneration

• alleles tend to be found in non-coding regions

• and/or typically only mildly affect expression

once you identify a putative gene mutation, how can you model the mutation’s effects to determine if it is indeed the disease-causing variant?

identify the impact of the mutation on global gene expression in cells

• RNA sequencing is one method

RNA-sequencing

• RNA → cDNA, which are then sequenced

• provides quantitative analysis of cell’s transcriptome

• can also detect rare splice variants

in situ hybridization

can tell when and where a particular gene is expressed by hybridizing a fluorescently labeled, singlestranded probe against complementary RNA sequences within cells

reporter genes

used to determine the pattern of a gene’s expression

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

• GFP is then controlled by the gene’s endogenous regulatory sequences

• ensures that GFP expression will match the normal expression patterns of the gene of interest

homologous recombination using ES cells

• DNA creation

1. gene sequence altered in cultured ES cells

2. DNA plamids introduced into ES cells w/ mutated DNA @ gene of interest, flanked by homologous DNA seq corresponding to ES cell

3. plasmid also contains selection marker (antibiotic resistance gene)

4. ES cells cultured in presence of antibiotic — only cells that take up plasmid + correctly swap out the DNA fragment will survive

5. pick + inject each surviving clone into blastocyst

6. blastocyst (black) inplanted into pseudopegnant mother (brown)

homologous recombination using ES cells

• results

1. results are chimeras — fur is white + brown (most likely to have mutation in germ cells

2. chimeras mated w/ normal mouse to generate F1s — sequences for the gene mutation to check successful transfer

3. F1s can mate to product mutant mice that have 2 copies of the mutant allele

CRISPR

1. Cas9 can induce double-stranded breaks into DNA, but it needs a guide RNA to target specific sequences

2. Cas9 + guide RNA are expressed in a cell of interest

3. guide RNA targets Cas9 to the gene of interest so it can induce a double stranded break

4. donor DNA is also added, and this contains a altered piece of DNA

5. homologous recombination swaps the wild-type gene for the donor gene, and enzymes that repair double stranded breaks finish the process

6. this can be done in the blastocyst, eliminating the need for ES cell based homologous recombination!

how can Cas9 actively a dormant gene/ turn off an active gene

by using a mutant form that can no longer cleave DNA

conditional knockouts (CKO)

a gene can be selectively disabled in a particular target tissue

CKO in mice

• mouse #1 = insert two pieces of DNA called LoxP sites into the flanking regions of the target gene

• mouse #2 = insert a piece of DNA encoding for the enzyme Cre recombinase — inserted down-stream of a gene promoter that is known to direct gene expression to a particular type of cell or tissue

• 1 + 2 mate— offspring that inherit the gene sequence flanked by LoxP sites, in addition to Cre-recombinase, will have excision of that gene is all cells where the gene promoter directing the expression of Cre recombinase is active