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Forward genetics
begin with a phenotype (mutant individual) and proceed to a gene that encodes a phenotype
Reverse genetics
begin with a gene of unknown function, induce mutation(s) in this gene, observe the effect of mutation(s) on the phenotype of organism
transgenic organism
n organism that has had its DNA altered by adding DNA from another species
Knock-out animal
modify gene sequence to create a loss of function allele
Knock-in animals
add an extra gene into the genome (from the same or different species)
Why make transgenic animals
determine the cellular/molecular function of a gene
model human conditions
analyze localization of specific proteins inside cells
identify a specific gene responsible for the mutant phenotype
rescue experiment
mutation in which gene causes (blank) phenotype?
Analyze gene expression patterns (reporter strains)
GFP is expressed in cells that normally express the gene
cells that normally express the gene are now labeled with GFP and can be visualized
Making transgenic (knock-in) mice by pronuclear injection
site of transgene insertion is random (and usually unknown), injected DNA will randomly integrate into one of the chromosomes
foreign DNA (many copies) injected into one of the pronuclei of a fertilized egg
If integration occurs before first mitosis
all cells of the embryos will carry the transgene
embryos are implanted in a surrogate mother
10-30% of embryos survive; only some will carry a copy of integrated transgene
Targeted mutagenesis
enables alternation of specific genomic sequences
Cas9
CRISPR-Associated protein (endonuclease)- enzyme, makes double stranded breaks in the DNA
sgRNA
single Guide RNA, single-stranded RNA= tacrRNA (binds endonuclease) + crRNA (binds target DNA sequence)
Genome editing by CRISPR/Cas9 step 1
the single guide RNA and Cas9 protein combine to form an effector complex
Genome editing by CRISPR/Cas9 step 2
effector complex associates with the PAM sequence, unwinds the DNA, and pairs with the complementary sequence on the DNA
PAM
protospacer Adjacent Motif (commonly NGG)
Genome editing by CRISPR/Cas9 step 3
the Cas protein cleaves the DNA
The accuracy and efficiency of genome editing are influenced by
double-stranded break repair pathways
non-homologous end joining (NHEJ)
Homology directed repair (HDR)
Non-homologous end joining (NHEJ)
can bring together any DNA ends prone to errors
Homology directed repair (HDR)
accurate repair of double-stranded breaks
requires a DNA template containing the desired edit
Gene therapy
genetic engineering of human somatic cells in order to treat genetic diseases
applying genetic tools to humans
Therapeutic gene
need to know what the disease-causing mutation is and understand the molecular mechanism of a disease (basic science research, model organism)
gene delivery method/vector
efficient delivery of nucleic acids to cells; protection from premature degradation
gene therapy for targeting…
specificity to a tissue/cell type of interest
Viral and nonviral vectors are used
to deliver DNA to specific cells and the nucleus (so it can be expressed!)
delivery to specific tissue in which the transgene is needed is key!
protect the DNA from degradation
Phase 1 clinical trial
focus on safety and the proper dose
Phase 2 clinical trial
focus on effectiveness and side effects
Phase 3 clinical trial
compare the new treatment to existing treatment
FDA reviews the results and can approve the drug for use on people outside the clinical trial
phase 4 clinical trial
treatment approved and available. Long-term effects are observed
look for side effects not seen earlier
study how well a new treatment works over a long period of time
In vivo
therapeutic drug is directly administered to the patient (e.g. injection into target organ)
ex vivo
patient-derived cells are genetically modified in a dish and then re-infused into the patient
