Transgenic Organisms Flashcards
Why study genes in a whole organism?
- Tissue cluture cells by definition are cells that have become immortalized due to some abnormality
- Can only give information on how a cell is affected, not how a whole organism is affected.
DNA delivery methods in higher eukaryotic cells
- Electroporation = similar to method for bacteria transformation by electroporation.
- Transfection = packing of DNA into liposomes and uptake of the liposomes by the cells
- Transduction = packaging DNA into a virus and infecting the cell with this virus. The virus has been genetically modified to prevent viral replication and transmission
- Bacterial infection = using the bacteria Agrobacterium tumefaciens to deliver DNA into plant cells
- Biolistics = coating gold or tungsten particles with DNA and ‘shooting’ the particles into cells
Adenovirus
- Contains a DNA genome
- Can infect dividing and non dividing cells
- Does NOT integrate into host chromosome (episomal)
- Vector on slide 5
Retrovirus
- Contains RNA genome
- Infects only dividing cells
- Integrates into host chromosome
Agrobacterium tumefaciens-mediated gene transfer
A. tumefaciens is a gram-negative bacteria that can infect dicot plants to form Crown Gall disease (tumor).
To be virulent, the bacteria must contain a Ti (tumor inducing) plasmid. These cells grow to form the tumor or all (slide 8) and this system can be harnessed to create a useful mechanism for transforming plants
Biolisticts
The 'Gene gun' was originally used to deliver DNA into oinion cells. Heavy metals (gold or tungsten) are coated with DNA and 'fired' into cells. Originally used in plants, but has also been used to deliver plasmid DNA and vaccines into animal subjects
Gene deliver technique advantages
- Retrovirus = Integrates into host cell genome providing stable gene expression
- Adenovirus = Can contain >30 kbp of non-viral DNA. Infects non-dividing and dividing cells
- Lipososmes = Non-pathogenic. Non-immunogenic. No limit to size of gene
- Bacterial infection = Efficient virulence and easily identifiable gene transfer (gall formation)
- Microinjection = Non-pathogenic. Non-immunogenic. No limit to size of gene
- Biolistics =Non-pathogenic. Non-immunogenic. No limit to size of gene
Gene deliver technique disadvantages
- Retrovirus = Random integration may cause insertional mutations. Only infects dividing cells
- Adenovirus = Does not provide long term gene expression. No integration (episomal). Immunogenic
- Lipososmes = Low transfection efficiency. Low rate of stable integration
- Bacterial infection = Random integration of DNA
- Microinjection = Limited to dermal and muscle tissue. Low rate of stable integration
- Biolistics = Limited to dermal tissue. Difficult to quality control.
Transgenic organisms
A ‘transgenic’ organism contains novel HERITABLE genetic material added by molecular cloning techniques.
Only applies to multicellular organisms.
Also referred to as GMO.
Transgenic organisms have many uses in medicine and agriculture:
1. Transgenic mice can be treated and used as models for disease
2. Transgenic plants can be used to create plants with advantageous features
3. Biopharming
Transgenic plant uses
- Herbicide resistance == “Round Up” resistance - EPSPS enzyme resistant to glycophosphate
- Pesticide resistance = ‘Bt’ corn - Bacillus thuringiensis protein is toxic to insects
- Add important nutrient or vaccine antigen = Vitamin A in golden rice. Hepatitis B antigen in potatoes
- Prevention/delay of spoilage = FLAVR SAVR tomato
FLAVR SAVR tomato
Non-GM tomato
1. Gene for polygalacturonase produces mRNA which is translated to the PG protein
2. Pectin is responsible in part for cell rigidity
3. PG cleaves pectin which results in loss of rigidity and increased softness of fruit
GM tomato
1. mRNA from antisense transgene lowers level of PG protein
2. Pectin cleavage is reduced and so the fruit remains for rigid. Other effects of ripening (color change from green to red) are unaffected
How to make a transgenic mouse
- Isolate fertilized egg
- Microinject eggs with transgene DNA
- Implant injected eggs into pseudopregnat foster female
- Birth and growth into adulthood
- Isolate tail DNA for PCR
- Analyze PCR products by gel electrophoresis
Verifying that the transgene has inserted into germ line
Just because a mouse has cells that contain the transgene DOES NOT meant that this mouse can pass the transgene on to its offspring.
The transgene must be in the germ cells to pass on to offspring. To determine this, mice bust be backcrossed and offspring examined (must be passed onto offspring)
Transgenic mouse as a disease model
TRAMP = transgenic Adenocarcinoma Mouse Prostate Model
- Microinject fertilized eggs with plasmid that contains prostate promoter
- Identify founder mice by backcrossing and PCR analysis
- Isolate liver, kidney and prostate RNA from founders and analyze by Northern blotting.
- Founder stains has prostate specific oncogene expression? = females have no ovarian tumors, prepubescent males have no prostate tumors, and adult males have large prostate tumors
Knocking-out gene function Method #1
Slide 19
