Molecular - transgenics

Question 1

What is a GMO?

Answer 1

Something that has taken up extracellular DNA

Question 2

GMO uses

Answer 2

Protein expression and harvest (spider goat, insulin); Host modification (pest or herbicide resistance, GH in salmon); Human health (unsat. oils in pigs or plants)

Question 3

Concerns with GMOs

Answer 3

Escape of transgenic into the wild, modifications that cause allergies, other side effects in humans

Question 4

Bt corn

Answer 4

Has gene from Bacillus thuringiensis, encodes delta endotoxin which kills insects that feed on the plant.

Question 5

Round up ready plants

Answer 5

Resistant to roundup aka glyphosate which inhibits enzyme needed to produce aromatic a.a.’s .

Question 6

Omega-3s

Answer 6

unsat. fatty acids with double bond after C 3. Lower LDL levels. Must come from diet. Found in fish (from algae)

Question 7

Omega-3 anti inflammatory

Answer 7

Partially block arachidonic acid (Omega-6); prostaglandins produce immune response

Question 8

Nutritionally important omega-3s

Answer 8

a-linolenic acid (ALA - 18:3), eicosapentaenoic acid (EPA - 20:5), docosahexaenoic acid (DHA - 22:6)

Question 9

Plasmid delivery

Answer 9

Used to insert genes into bacteria (need to use cDNA if using eukaryote genes - bac can’t do introns)

Question 10

pCAGGS expression vector

Answer 10

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

Question 11

Ti Plasmid

Answer 11

“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

Question 12

Ti Plasmid uses

Answer 12

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

Question 13

Other method to insert Ti plasmid

Answer 13

Vacuum delivery - entry backwards through stomata. Another is gene gun and shoot into embryo etc.

Question 14

Gene trapping premise

Answer 14

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

Question 15

Gene trap details from slides

Answer 15

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.

Question 16

Uses of gene trapping

Answer 16

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)

Question 17

SA

Answer 17

Splice acceptor at 3’ end of intron. interrupts normal splicing and causes the downstream vector sequence to be transcribed.

Question 18

Selectable marker

Answer 18

antibiotic or other. Used to identify insertion worked. Ex. Neo

Question 19

pA

Answer 19

polyadenylation site; separates exons by stopping translation

Question 20

PGK

Answer 20

Strong promoter in embryonic stem cells (ESC) to restart transcription after the PolyA section of the first half of the gene trap

Question 21

BtK

Answer 21

Brutons tyrosine kinase exon 1.

Question 22

SD

Answer 22

Splice donor - allows the other half of the trap to bind to the next exon

Question 23

BLAST

Answer 23

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)

Question 24

Larger gaps in BLAST

Answer 24

still count as one penalty in scoring, attributed to one insertion/deletion event

Question 25

Modified gene trap

Answer 25

Use as a delivery system. Replace the reporter gene with gene of interest. Ex. insert desaturase into cell, nucleus inserted into oocyte

Question 26

Gene trap applications

Answer 26

Insert desaturase into pigs, the express omega-3s in tissues. Knockout galactose-a1-3 galactose moiety on porcine glycoproteins, eliminate response to organ transplant