LECTURE 10 (transgenics)

Question 1

What are the key three steps in transgenics?

Answer 1

• introduce the transgene
• select for the trangenics events
• express the transgene (where they go in a cell)

Question 2

How do we get from a gene to a phenotype(two ways) which one is the way used in transgenics?

Answer 2

• forward genetics; identify variation (phenotype) isolate mutants and identify the phenotype alteration, they you know which are the genes that work.
• reverse genetics: you start with an isolated gene and then you create mutations and observe the effect on the phenotype. this is used in transgenics

Question 3

define transgenics

Answer 3

the creation of a new organism whose genome is altered by the introduction of foreign DNA.

Question 4

What are the main aims of introducing a transgene into an organism?

Answer 4

• mutation
• over-express certain genes and get more substrate produced
• down-regulation to turn down the expression of certain genes
• ablation: complete elimination of a gene (gene K.O)
• miss-expression: make transgenics where gene is expressed at different times

Question 5

what is the ablation of a gene?

Answer 5

the complete elimination of a gene through transgenesis.

Question 6

How can a transgene be inserted into a cell?

Answer 6

Artificially:

• microinjection
• biolistics
• electroporation
• PEG/CaCl2

Naturally: via bacteria

Question 7

What is microinjection?

Answer 7

physically inject DNA into the nucleus of an organism e.g in mosquitoes can be done e.g making a transgenic mouse

Question 8

How’s a transgenic mouse done?

Answer 8

You get a previously fertilized egg from a female, insert the transgene into the male protonucleous, then inserted into a pseudopregnant mouse and check the offspring because some of them will have the transgene

Question 9

PEG/CaCl2 as a method for trangenics why?

Answer 9

these compounds make temporal wholes in the nucleus membrane allowing other genes to get into it.

Question 10

What is biolistics?

Answer 10

This is another artificial way of making transgenics. you cover metal particles with NA and the gene you wanna insert, then target a tissue, they’re at such a high speed that get into the nucleus.

Question 11

What is electroporation?

Answer 11

It’s a method to make transgenics. It’s a machine similar to a spectrophotometer but it does not measure the UV you put your cells into a cuvette then apply a very high voltage in the machines which induces the formation of pores in the membranes and the DNA can get into the cells. It’s used with E.coli.

Question 12

Are transgenics organisms always artificial? Is it always bad?

Answer 12

No, this happens naturally in life. e.g Agrobacterium tumefaciend. It’s not always bad in fact it has been found that sweet potatoes contain the T-DNA from agrobacterium

Question 13

Describe the case of Agrobacterium tumefaciend.

Answer 13

This is a bacterium that infects plants. it has a plasmid TI plasmid (tumor inducent plasmid) which contains T-DNA. This T-DNA encodes the biosynthesis of sugars which is imp for the bacteria. when this bacterium infects a plant, T-DNA gets integrated into the genome and the plant will develop a tumor plus there’s biosynthesis of sugars good for the bact

Question 14

What is genome editing?

Answer 14

Change the genome of interest to obtain a desired alteration in the target gene.

Question 15

Difference between cloning and transgenes?

Answer 15

Cloning: obtain progeny that is genetically identical to original plant or animal
Trangenics: introduction of foreign DNA into an organism.

Question 16

What are mosaics animals?

Answer 16

= chimaeras

animals that have only expressed certain gene in some cells and not in other.

Question 17

What’s a selectable marker? (3)

Answer 17

• a gene that confers antibiotic resistance
• a gene that confers prototrophy to auxotrophic mutants
• a gene that confers the ability to grow under certain conditions.

Question 18

The use of transgenic organisms must be monitored because…

Answer 18

the law tells us to do so and it is good practice due to the possibility of hazards resulting from the creation of new variants

Question 19

What is a cassette in a plasmid?

Answer 19

Region that contains an specific recombination site.

Question 20

Why is Arabidopsis used instead of tobacco plants for transgenics?

Answer 20

because you can simply dip the flowers of arabidopsis into a medium with agrobacterium and will eventually produce some seed that will be transgenic (faster).

Question 21

How can we make transgenic plants?

Answer 21

Taking advantage of agrobacterium deleting the tumor inducing genes of T-DNA and replacing by our gene of interest, then either dipping the flowers of Arabidopsis or making cuts in tobacco plants grow with the bact.

Question 22

applications of alterations of homologous genes in transgenic organisms:

Answer 22

• increased production of biotechnologically important enzymes in soil fungi
• mprove the chances of xenotransplantation by interfering with the antigenicity of pig organs to be used in human transplants
• alteration of hormone levels to improve and speed up growth of livestock

Question 23

applications of over- expression of heterologous genes in transgenic organisms

Answer 23

• resistance to pests and disease
• production of proteins of pharmacological use
• resistance to herbicides in crops

Question 24

What are TALEs and why are they used for trangenes.

Answer 24

they are proteins made up of repeats that in nature bind to DNA seq. these repeats determines which DNA they bind so we can make engineered TALEs to recognize the seq we want.

Question 25

What is the CRISPR–cas9 system? Why is it used in genome editing?

Answer 25

It’s an acquired immune response in bacteria. when a bacteria is infected by a phage CAS proteins degrade the DNA and introduce it into the CRISPR array and the next time the bact is infected by this type of phage they’ll be immune. we can exploit this to modify the target genes.