Molecular - transgenics Flashcards
(26 cards)
What is a GMO?
Something that has taken up extracellular DNA
GMO uses
Protein expression and harvest (spider goat, insulin); Host modification (pest or herbicide resistance, GH in salmon); Human health (unsat. oils in pigs or plants)
Concerns with GMOs
Escape of transgenic into the wild, modifications that cause allergies, other side effects in humans
Bt corn
Has gene from Bacillus thuringiensis, encodes delta endotoxin which kills insects that feed on the plant.
Round up ready plants
Resistant to roundup aka glyphosate which inhibits enzyme needed to produce aromatic a.a.’s .
Omega-3s
unsat. fatty acids with double bond after C 3. Lower LDL levels. Must come from diet. Found in fish (from algae)
Omega-3 anti inflammatory
Partially block arachidonic acid (Omega-6); prostaglandins produce immune response
Nutritionally important omega-3s
a-linolenic acid (ALA - 18:3), eicosapentaenoic acid (EPA - 20:5), docosahexaenoic acid (DHA - 22:6)
Plasmid delivery
Used to insert genes into bacteria (need to use cDNA if using eukaryote genes - bac can’t do introns)
pCAGGS expression vector
Neo (antibiotic resistance), chicken B-actin/rabbit B-globin hybrid promoter (AG), human CMV-IE enhancer, SV40 origin and Poly A site, E. coli lac operon promoter. Can infect bacteria or mammalian cells and express gene
Ti Plasmid
“Tumor inducing”. Agrobacterium infects plants, and includes plant hormone genes in T-DNA (transfer DNA) region which causes growth of a tumor that bacteria use as food. Plasmid gets integrated in genome
Ti Plasmid uses
Standard to infect plants. Can replace the plant hormone genes (in T-DNA region) with anything so you can express and harvest proteins from plants
Other method to insert Ti plasmid
Vacuum delivery - entry backwards through stomata. Another is gene gun and shoot into embryo etc.
Gene trapping premise
Retroviral integration into genome in an intron. It has a promoterless reporter (neo) and a downstream promoter which helps determine where it inserted. It inactivates the gene and the trap can then be used to add primers to sequence the transcript
Gene trap details from slides
Two parts: Part 1:SA, selectable marker, pA. Transcribed in response to normal gene promoter. Part 2: PGK reinitiates transcription. Btk creates sequence tag. SD is splice donor. Fuses to end of next exon.
Uses of gene trapping
We are generating sequence and target introns, separate exons, without gene transcription. high throughput method to make cell lines with single gene knockout. We can recover sequence, compare to BLAST, locate position in genome (and which gene we knocked out)
SA
Splice acceptor at 3’ end of intron. interrupts normal splicing and causes the downstream vector sequence to be transcribed.
Selectable marker
antibiotic or other. Used to identify insertion worked. Ex. Neo
pA
polyadenylation site; separates exons by stopping translation
PGK
Strong promoter in embryonic stem cells (ESC) to restart transcription after the PolyA section of the first half of the gene trap
BtK
Brutons tyrosine kinase exon 1.
SD
Splice donor - allows the other half of the trap to bind to the next exon
BLAST
Basic local alignment Search Tool. Seeding with small portion of sequence. Find small sample (search DNA or protein). Extend in both directions, score matches and penalize for adding gaps/mismatches (but can make gap to compensate for a possible “insertion”). Get high scoring segment (HSP)
Larger gaps in BLAST
still count as one penalty in scoring, attributed to one insertion/deletion event
