H5 Jarvis- Medical and agricultural applications of gene technology

what is the difference between transient and stable transformation?

what are the methods of gene transformation?

• CaPO₄ precipitation

• electroporation

• microinjection

• microprojectile bombardment

• viral vectors (for mammals)

• agrobacterium-mediated (for plants)

what is the process, pros and cons of CaPO₄ precipitation as a method of gene transformation?

advantages

• quick, cheap and simple

• not vector dependent

• can be used for transient or stable expression (this requires a selectable marker, to select for cells where the plasmid DNA has integrated)

limitations

• essentially only used for mammalian cell lines

what is the process, pros and cons of electroporation as a method of gene transformation?

advantages

• quick

• not vector dependent

• used for bacteria, yeast, plant protoplasts, and mammalian cells (e.g. embryonic stem cells used when making knockout mice)

• can be used for transient or stable expression (random or specific)

limitations

• equipment is quite expensive

• can only be used for single cells (so to study a multicellular organisms it must be able to regenerate from a single cell, e.g. when making knockout mice, transformed stem cells are injected into a blastocyst, which is implanted into a surrogate mother)

what is the process, pros and cons of microinjection as a method of gene transformation?

advantages

• not vector dependent

• allows stable integration (but usually random)

limitations

• expensive + requires skill

• very labour-intensive

• random stable insertion point (can’t be used for knockout mice)

• not very useful for plant cells

what is the process, pros and cons of microprojectile bombardment as a method of gene transformation?

advantages

• quick

• can in principle be used on any tissue, including plants

• not vector dependent

• can be used for transient or stable expression

• can deliver DNA to organelles (usually chloroplasts)

limitations

• equipment is expensive

• best suited for use with ‘robust’ cells (usually plants)

what is the process, pros and cons of viral vectors (for mammals) as a method of gene transformation?

advantages

• effective delivery of DNA to cells or intact organisms

• applicable to many systems owing to diversity of viruses

• can be used for transient (e.g. adenovirus) or stable expression (e.g. lentivirus)

limitations

• highly vector dependent

• integrating vectors may activate cellular oncogenes due to random integration

• non-integrating vectors offer less stable expression, particularly in dividing cells

what is the process, pros and cons of agrobacterium (for plants) as a method of gene transformation?

advantages

• widely and routinely used- many vector options available

• enables stable integration (needs a selectable marker)

limitations

• limited host range (good for dicots, less for gymnosperms and monocots)

• can be time-consuming (depending on species)

• site of integration in genome is random

how can designer nucleases be used for genome editing?

what are some examples of agricultural and medicinal applications of gene modification?

agricultural:

• deactivation of polygalacturonase (degrades pectin, so tomatoes must be harvested earlier = less flavourful) by RNAi

• glyphosphate herbicide resistant crops (to use bacterial version of amino acid synthesis enzyme that is targeted by glyphosphate)

• insect resistant crops by expressing Bt toxin

• golden rice producing vitamin A to combact deficiencies

medicinal:

• hormone production eg. insulin/human growth hormone using bacteria

• vaccine production eg. Hep B using yeast

• gene therapy- somatic vs germline eg. SCID, cystic fibrosis, sickle cell disease