Ch 1.3 Flashcards
Genetic engineering in plants
Transgenic plants: Using Ti plasmid as a vector in plants The VIR (virulent) region of the plasmid allows entrance into plant cells. Here a gene conferring resistance to herbicide is introduced into plant cells. Select in the presence of herbicide herb.res is ectopic insertion (the orange bit). we don’t know where it’s gonna go. (that gene is gonna be inserted into the genome but we don’t know where it’s going to go
Examples of transgenic plants
Rice that produces vitamin A. Genetically engineered corn, cotton and other crop plants express such insecticidal proteins. Soybeans producing more monosaturated fatty acids. Soybeans producing compounds useful as lubricants, paint, and plasticizers. Transgenic plants producing vaccine products. Easily to grow and to administer.
How to detect the presence of the transgene
Use a reporter gene (promoter like LacZ, GFP), phenotype, Molculurar methods: run PCR for gene of interest
Gene function - knockouts Vs. Conditional knockouts
1) Modify form of gene linked to genes for neomycin resistance and sensitivity to glanciclovir. 2) Deliver modified gene into embryonic stem (ES) cells. Recombinant ES cells are recovered that contain a disrupted or knockout version of the gene of interest CONDITIONAL: Knockout is more precise. Cre-loxP recombinase system The gene to be KO is introduced flanked by loxP sites You can activate an enzyme (Cre recombinase) that cuts any sequenced flanked by loxP sites.
RNA interference
to switch off deliver in a double strand RNA into fly cell so it will think its weird and attack. So make the RNA complementary to the gene of interest. So attacked by dicer so dicer chops up into segments recognized by RISC that makes then single. a complex also binds to it. Now they have complimentary single strands that bind with the gene of interest (mrna) and then this gets chopped off because its now double stranded RNA again. RNA INTERFERENCE Double stranded RNA (dsRNA) deliver into cell/ organism. dsRNA is cleaved by Dicer. The short dsRNA can inhibit expression (RNAi) of a gene with complementary sequence by the action of RNA-induced silencing complex (RISC). A specific gene can be targeted and shutdown by delivering dsRNA.
CRISPER
Cluster of Regularly Interspaced Short Palindromic Repeats. 3 steps for immunity: adaption, crRNA biogenetics, interference. 1. genome specific crRNA sequence, 2)genomic specific trcrRNA-crRNA chimera, 3)a sequence motif like PAM. Crisper sends cas9 to cut a specific RNA sequence it saved thus deleting or inserting certain sequences in the dNA (By Non-homologous end joining (NHEJ) Leads to insertion/ deletion)
Gene therapy
Adding a normal copy of a gene to the genome of an individual carrying a defective copy.
Somatic gene therapy
Remove cells from patient with the disease and make them transgenic with the introduction of the wild-type gene. Reintroduce into patient, immuno deficiencies.
Germ-line gene therapy
Correction of the disease and transmission of the normal genotype to the progeny –mitochondrial diseases –CRISPR editing? EX: Transfer of spindle chromosomal complex between donor and recipient cells. Tested in humans as a viable approach to avoid transmission of serious mitochondrial diseases. The technique isolates and transplants the chromosomes (nuclear genetic material) from a patient’s unfertilized oocyte into the cytoplasm of another enucleated egg, containing healthy mtDNA. progeny also cured
Viral vectors in somatic human gene therapy
Retrovirus: Attacks proliferating cells (many disorders attack cells that rarely divide) Can integrate into patient genome (insertional mutagenesis)
Adenovirus: Attacks non dividing cells Does not integrate into patient genome (repeated treatments)
Reproductive vs. therapeutic cloning.
Reproductive cloning is the creation of an animal. Most serious concerns are developmental defects. Therapeutic cloning is the creation of early stage embryos to harvest stem cells for treatment of disease Method: Nuclear transfer (replace the nucleus of an egg with the nucleus from an adult cell).
Four factors made ADA-SCID a good candidate for gene therapy
–Gene was cloned and well characterized
–Blood cells can be easily obtained from patients and reintroduced after adding functional copies of the ADA gene.
–Even small amounts of functional ADA will restore partial immune function
–Overproduction of ADA has no toxic effects
