What are GMOs?
- Selective breeding and cross-breeding in crops
- Modern biotechnology, especially “genetic engineering” allows qualitative change in way genetic manipulation and breeding can be carried out in agricultural species
- Can identify and select genes from one organism and transfer them into another organism (allows recipient to do something it couldn’t do before)
How do you make transgenic animals by microinjection?
- Haploid oocyte (egg) x haploid sperm
- Wash fertilized egg out of females oviduct
- Diploid fertilized egg with male and female pronuclei (which eventually fuse to form the zygonucleus)
- Before this fusion occurs, foreign DNA is microinjected into a pronucleus (either male or female, usually male because it is bigger)
- To inject DNA, egg is held by a suction pipette and gene of interest found in DNA solution is injected into egg
- Eggs are implanted into a pseudopregnant female mouse
- Offspring are born and DNA is analysed to determind which has the transfene present
Why is it important to add DNA before the pronuclei fuse?
- DNA repair enzymes will still take up DNA at this stage
- Need to flush out eggs shortly after fertilization to ensure the pronuclei have not yet fused
How do you prepare fertilized eggs for microinjection?
- Make small drops of medium on tissue culture dish
- Flood with paraffin oil (prevents evaporation of medium)
- Equilibrate in incubator
- Add embryos to microdrops (embryos are lined up between two air bubbles in micropipette)
What type of microscopy shows injection has been successful?
- Interference microscopy (has better contrast than other types of microscopy)
Why is microinjection not a desirable technique to use?
- Expensive
- Very inefficient (embryos need to successfully be injected, implanted, need live born, and transgenic progeny)
What does totipotent mean? What cells are totipotent?
- Cell is able to form any cell in the body
- Zygotes are totipotent
What does pluripotent mean? What cells structures are pluripotent?
- Variety of possibilities for cells, but not totally competent
- Morula (solid ball of cells) and blastocyst (hollow ball of cells) are pluripotent
What are the three categories that stem cells can divide into? What are some structures that these categories give rise to?
- Ectoderm (brain, skin cells)
- Mesoderm (bone marrow, muscles)
- Endoderm (liver, GI tract)
What does multipotent mean?
- Smaller variety for cells
- Can still overlap into other categories of cells (e.g., ectoderm cells may still become mesoderm cells)
What does progenitor mean?
- Cell is specialized and committed to one type of cell
Describe in vitro differentiation of embryonic stem cells
- Embryonic stem cells are pluripotent
- Embryoid bodies life off media and will form balls of cells called blastocysts
- This gives rise to mesoderm, ectoderm, and endoderm cell types (can stain with ABs to determine cell type)
How can you create transgenic mice with embryonic stem cell technology?
- Mate male and female mice
- Isolate blastocyst (allow embryo to continue to blastocyst stage)
- Early blastocyst is when gene transfer occurs, flush it out of female
- Transfer to petri dish
- Inner cell mass will be on dish (eventually will give rise to organs)
- Remove inner cell mass
- Dissociate with trypsin (enzyme)
- Plate in petri dish with feeder layer of cells
Why plate on feeder layer?
- Want to keep cells in primitive state (keep them from touching plastic, otherwise they will grow up and round out)
- Certain factors will keep the cells in primitive state
- Fibroblasts’ DNA is zapped with gamma rays to prevent cell division
- These cells can still provide nutrients for ESCs to grow
- Help grow but don’t contaminate because they cannot divide
After embryonic stem cells have been plated and cultured in petri dish, what are the next steps in creating transgenic mice?
- Cells are injected into cavity of blastocyst from a DIFFERENT mouse strain
- Hope that some ESC genetic modifications end up in gonads so they will be transferred to offspring (but have no control over which organs are formed by ESCs)
- Breed brown mouse with chimeric mouse - if one offspring is beige (like original mouse that donated blastocyst) then it must contain (heterozygous) genetic modifications in all nucleated cells
- Gene may not be expressed at all though
Why are transgenic animals from ESCs desirable?
- Easy, cheap
- Less precise equipment needed
How can you clone transgenic animals using nuclear transfer?
- Cells in cell cycle will enter G0 if growth factor supply is blocked
- Donor G0 cells are injected into enucleate (pronucleus removed) oocytes under zona pellucida
- Electric pulse sent through unfertilized cell, diploid nucleus fuses with cell
- Now have “renucleated” oocyte
- This oocyte is genetically identical to donor cell and is “fertilized”
- Transferred to foster ewe
Give a generic overview of steps in nuclear transfer cloning
- Mammary cells cultured
- Starve G0
- Add to enucleate oocyte
- Implant in foster mother
- “Dolly” (cloned sheep) is born
Is the cloned offspring genetically identical to its “parent”?
No.
- Nuclear DNA is identical, but mitochondrial DNA is not
What are some conclusions that can be drawn from the Dolly study?
- Suggests the existence of “oocyte cytoplasmic factors” critical for reprogramming of the quiescent donor cell nuclei
- Suggests that nuclei form a wide range of cell types could be used as donors
- “First mammal to develop from a cell derived from an adult tissue”
- “During development and differentiation of the mammary cell, there was no irreversible modification of genetic information required for development to term”
What are some applications of transgenesis in animals?
- Introduction of growth-related genes
- Deletion of disease-related genes
- Introduction of immune-enhancing genes
- Mammary gland-specific gene expression **
- Introduction of metabolic pathway genes
- Blood-specific gene expression
How can we use transgenic sheep as a pharmaceutical tool?
- Pharmaceutical proteins can be produced in the milk of sheep
- Use animal as “bioreactor”
- Gene is injected into pronucleus as part of plasmid
- Use tissue-specific promotor (only expressed in mammary gland - allows for mammary gland specific expression)
- Foreign protein is expressed in milk during normal lactation cycle
- Foreign protein can be collected and purified by conventional biochemical techniques
Why were strategies to express proteins in transgenic animals developed?
- Levels of production by conventional prokaryotic expression systems were too low or were toxic to bacterial host (economically non-viable)
- Isolation from naturally-occurring sources were too expensive or posed a biohazard risk
- The recombinant protein produced by prokaryotic expression systems was not biologically active due to lack of eukaryotic modification (e.g., glycolysis)
- After initial high investment costs of developing transgenic animals, the actual cost of making the recombinant protein are much lower than the continuing high costs of large scale prokaryotic cell culture (high upfront cost, low continuing cost)
Why are mammary glands selected as site for production of novel proteins?
- Secrete substantial quantities of proteins into milk
- Milk can be collected daily
- Relative few major proteins to contaminate recombinant protein
- Recombinant protein is “isolated” from animal and thus avoids potential side-effects on animal
- Milk is sterile secreted material that is relatively predictable in its composition
