Lecture 28: Genetically modified Organisms

Question 1

Examples of GMOs

Answer 1

• genetically modified Salmon:
  
  • growth hormone
  • Pout AFP (antifreeze gene)
• Transgenic mice
  
  • rat growth Hormone - controlled by Metallothionein gene promoter

Question 2

making transgenic mice process (general)

Answer 2

Process

1. transgene construction
   
   • promoter (+cis regulatory elements)
   • cDNA - gene of interest
     
     • add polyadenylation signal so it becomes stable mRNA
2. fertilized egg with two hapload pronuclei
3. microinjetion of DNA (transgene) into male pronucleus

• non-homologous recombination (double strand-break repair) before 1st cell division

1. forms dipload zygotic nucleus
2. implant nucleus into foster mother
3. analyze mouse pups
4. test for transgene

• Founder (Fo) = Hemizygous: transgenic mouse

Question 3

Inducible transgene expression process: Tetracycline

Answer 3

• Tetracycline (tet): normally negatively controlled
  
  • then the Tetracycline binds to repressor so that the repressor is deactivated and the gene is expressed
• Tetracycline can be modified so that the repressor becomes an activator
  
  • presence of tetracycline deactivates the repressor (which is an activator) and stops transcription

Question 4

making a knockout mouse process

Answer 4

• requires selectable marker and two homologous flanking sequences
• possible products (eg neo resistance gene)
  
  1. no integration: killed by neomycin
  2. homologous recombination: resistant to neomycin and ganciclovir
     
     • inserts neo resistance gene but not the thymidine kinase gene
  3. non-homologous recombination: resistant to neomycin and killed by ganciclovir
     
     • inserts neoresistance gene and the thymidine kinase gene
• process:
  
  1. embryo from brown mice
  2. isolate embryonic stem (ES) cells
  3. transfect cells with vector
  4. select for homologous recombination by growing in neomycin and ganciclovir
  5. inject selected ES cells into blastocyst from mice with black fur
  6. impant into mother
  7. chimeric mouse (with brown and black fur): incorporated transgenic cells in them
  8. breed them out so that there will be homozygosity

Question 5

animal clones

Answer 5

• nucleus from terminally differentiated cells retain genetic info for whole animal (totipotent)
• introdcued nucleus - can be reprogrammed (epigenetics, chromatin structure, etc.)
• clone problems
  
  • shorter telomeres
• Process
  
  1. donor adult cells grown in culture (G1)
  2. donor adult cells deprived of nutrients - put into G0 - no cell division
  3. egg cell at metaphase II - discard chromosomes
  4. inject donor cells into egg cell
  5. electrick shock - causes fusion
  6. donor cell nucleus and mitochrondria with egg cytoplasm and mitochrondria
  7. forms embryo
  8. transfer to foster mom
  9. cloned lamb

Question 6

stem cells

Answer 6

• pluripotent - can form all 3 germ layers
  
  • good for replacing tissues
  • 3 layers
    
    • gut endotheium (endoderm)
    • muscle (mesoderm)
    • Neural/squamous epithelium (ectoderm)
• treating deficiencies process
  
  1. remove nucleus of human egg
  2. fuse nucleus of patient’s cell into empty egg
  3. grow to blastocyst stage
  4. harvest and culture inner pluripotent stem cells
  5. differentiation signals to form different tissues

Question 7

synthetic biology

Answer 7

• creation of bacterial cell with chemically synthesized genome
  
  • tried Mycoplasma gentialium - but grows too slowly so gave up
  • synthesized Mycoplasma mycoides - more complex (just over 1,000,000 bp) but faster growing
  • process:
    
    1. oligonucleotide synthesizer
    2. oligonucleotides (20x100nt=1kb)
    3. 1080 bp cassettes (10x1kb=10kb) - 80 bp overlap
    4. 10,080 bp cassettes (11x10kb=100kb) 80 bp overlap
    5. 1,080,000 bp (11x100kb=1000kb) 80 bp overlap
• leaves watermarks