Transgenic Talk


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Transgenic Talk

  1. 1. Transgenic mice: generation and husbandry
  2. 2. Transgenic vs. “knock-out” <ul><li>Transgenic: an organism that has had DNA introduced into one or more of its cells artificially </li></ul><ul><li>“ transgenic”: DNA is integrated in a random fashion by injecting it into the pronucleus of a fertilized ovum </li></ul><ul><ul><li>Random (approx.. 10% disrupt an endogenous gene important for normal development) </li></ul></ul><ul><ul><li>multiple copies </li></ul></ul>
  3. 3. Transgenic vs. “knock-out” <ul><li>Transgenic: an organism that has had DNA introduced into one or more of its cells artificially </li></ul><ul><li>“ transgenic”: DNA is integrated in a random fashion by injecting it into the pronucleus of a fertilized ovum </li></ul><ul><ul><li>Random (approx.. 10% disrupt an endogenous gene important for normal development) </li></ul></ul><ul><ul><li>multiple copies </li></ul></ul>
  4. 4. Transgenic vs. “knock-out” <ul><li>“ knockout”: DNA is introduced first into embryonic stem (ES) cells. ES cells that have undergone homologous recombination are identified and injected into a 4 day old mouse embryo - a blastocyst </li></ul><ul><ul><li>targeted insertion </li></ul></ul>
  5. 6. Transgenic production <ul><li>Transgenic mice are often generated to </li></ul><ul><li>1. characterize the ability of a promoter to direct tissue-specific gene expression </li></ul><ul><ul><li>e.g. a promoter can be attached to a reporter gene such as LacZ or GFP </li></ul></ul><ul><ul><li>2. examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals </li></ul></ul>
  6. 7. Brinster's growth hormone mouse
  7. 8. Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene. Neural structures expressing the reporter transgene are dark blue-green. (Dr. Anne Calof)
  8. 9. GFP transgenic mouse (Nagy) Tail tip 9.5 day embryos - GFP and wt
  9. 10. GFP transgenic mouse (Nagy)
  10. 12. Planning a Transgenic production mouse colony <ul><li>Mouse strain - popular </li></ul><ul><li>Colony size </li></ul><ul><ul><li>typical injection 200 embryos (7-10 females s.o.) </li></ul></ul><ul><ul><li>Superovulation efficiency </li></ul></ul><ul><ul><li>Parenting suitability </li></ul></ul><ul><ul><li>Pseudo-pregs </li></ul></ul>
  11. 14. Injecting fertilized eggs <ul><li>The eggs are harvested 0.5 dpc (superovulated or natural matings) </li></ul><ul><li>The DNA is usually injected into the male pronucleus </li></ul><ul><li>The eggs can be transferred the same day or the next (2-cell) into pseudopregnant female oviducts </li></ul>
  12. 15. Pronuclear injection
  13. 16. Implantation of 1 or 2 cell embryos <ul><li>The injected eggs are implanted the same day or are incubated overnight and implanted the next day </li></ul><ul><li>Injected eggs are transferred to the oviduct of a 0.5 dpc pseudopregnant female </li></ul>
  14. 17. Implanting 1(or 2) cell embryos 1 2
  15. 18. Implanting 1(or 2) cell embryos (cont.) 3
  16. 19. Pseudopregnant females and vasectomized males <ul><li>Female mice can be tricked into thinking they are pregnant </li></ul><ul><li>A mouse in estrus is mated with a vasectomized male </li></ul><ul><li>pseudopregnancy </li></ul><ul><li>If eggs (blastocysts) implanted will become truly pregnant and will give birth to live offspring </li></ul>
  17. 20. Vasectomizing 1 2
  18. 21. Breeding Tg founders <ul><li>Individually backcrossed to the strain of choice </li></ul><ul><li>DO NOT intercross different founders - each founder results from a separate RANDOM transgene integration even </li></ul>
  19. 22. Transgenic mice as tools <ul><li>Study gene function </li></ul><ul><ul><li>Many human diseases can be modeled by introducing the same mutation into the mouse. Intact organism provides a more complete and physiologically relevant picture of a transgene's function than in vitro testing </li></ul></ul><ul><li>Drug testing </li></ul>
  20. 23. Transgenic mice as tools <ul><li>Polio virus receptor </li></ul><ul><li>Normal mice can't be infected with polio virus. They lack the cell-surface molecule that, in humans, serves as the receptor for the virus. </li></ul><ul><li>Tg mice expressing the human gene for the receptor can be infected by polio virus and even develop paralysis and other pathological changes characteristic of the disease in humans </li></ul>
  21. 26. Vector design <ul><li>Recombinant DNA methods: Simple KO </li></ul><ul><ul><li>Structural gene desired (e.g. insulin gene) to be &quot;knocked out&quot; is replaced partly or completely by a positive selection marker. (knock out function!) </li></ul></ul><ul><ul><li>Vector DNA to enable the molecules to be inserted into host DNA molecules </li></ul></ul>
  22. 27. Typical KO vector *tk:thymidine kinase
  23. 28. Embryonic stem cells <ul><li>Harvested from the inner cell mass of mouse blastocysts </li></ul><ul><li>Grown in culture and retain their full potential to produce all the cells of the mature animal, including its gametes </li></ul>
  24. 29. ES cells growing in culture
  25. 30. ES cells are transformed <ul><li>Cultured ES cells are exposed to the vector </li></ul><ul><li>Electroporation punched holes in the walls of the ES cells </li></ul><ul><li>Vector in solution flows into the ES cells </li></ul><ul><li>The cells that don't die are selected for transformation using the positive selection marker </li></ul><ul><li>Randomly inserted vectors will be killed by gancyclovir </li></ul>
  26. 32. Successfully transformed ES cells are injected into blastocysts
  27. 33. Implantation of blastocysts <ul><li>The blastocysts are left to rest for a couple of hours </li></ul><ul><li>Expanded blastocysts are transferred to the uterine horn of a 2.5 dpc pseudopregnant female </li></ul><ul><li>Max. 1/3 of transferred blasts will develop into healthy pups </li></ul>
  28. 34. Implanting blastocysts 1 2
  29. 35. Implanting blastocysts (cont.) 3 4
  30. 36. Littermates Black mouse - no apparent ES cell contribution Chimeric founder - strong ES cell contribution Chimeric founder - weaker ES cell contribution
  31. 37. Chimeric mouse
  32. 38. Testing the offspring <ul><li>A small piece of tissue - tail or ear - is examined for the desired gene </li></ul><ul><li>10-20% will have it and they will be heterozygous for the gene </li></ul>
  33. 39. Breeding Chimeras (knock-out founder) <ul><li>Chimera - the founder </li></ul><ul><ul><li>germ-line transmission - usually the ES cells are derived from a 129 strain (agouti or white colour) and the ES cells are injected into a C57Bl/6 blastocyst (black). The more that the ES cells contribute to the genome of the mouse, the more the coat colour will be agouti. The chimera mouse is usually “tiger” striped. </li></ul></ul>
  34. 40. Breeding Chimeras (knock-out founder)cont <ul><li>Males that are 40% to 100% based on agouti coat colour should be bred </li></ul><ul><li>Females should not be bred (low incidence of success) ES cells are male. </li></ul><ul><li>Breed aggressively- rotate females through male's cage. If the male produces more than 6 litters without transmitting, not likely to go germline and should be sac'ed </li></ul>
  35. 41. Knock-out mice as tools <ul><li>If the replacement gene is nonfunctional (null allele), mating of the heterozygous will produce a strain of &quot;knock-outs' homozygous for the nonfunctional gene (both copies are knocked-out </li></ul><ul><ul><li>Find out if the gene is indispensable (suprisingly many are not!) </li></ul></ul><ul><ul><li>&quot;pleiotropic&quot; expression in different tissues in different ways and at different times in development </li></ul></ul>
  36. 42. Breeding Transgenics <ul><li>Most transgenics are bred onto a C57Bl/6 background </li></ul><ul><ul><li>standard </li></ul></ul><ul><li>BL/6 breeding information </li></ul><ul><ul><li>mate 6-8 weeks for best reproductive performance </li></ul></ul><ul><ul><li>replace males when 1 year old </li></ul></ul>
  37. 43. Breeding Transgenics (cont.) <ul><ul><li>Replace females after 6 litters or at 6 months of age </li></ul></ul><ul><ul><li>quick breeding - 1 founder male: 2 females </li></ul></ul><ul><ul><li>rotation of females through male cage </li></ul></ul><ul><li>Common problems: </li></ul><ul><ul><li>female not good mother, check for milk - give auntie </li></ul></ul><ul><ul><li>male cannibalizing litter </li></ul></ul><ul><ul><li>fighting (separate) Do not “reunite” males </li></ul></ul>
  38. 44. Breeding Transgenics (cont) <ul><li>Stick to schedules or be overwhelmed </li></ul><ul><ul><li>strict records (birth, ID, parents) </li></ul></ul><ul><ul><li>ID pups </li></ul></ul><ul><ul><li>tail tip or collect ear tissue at 2 weeks </li></ul></ul><ul><ul><li>try to genotype before weaning </li></ul></ul><ul><ul><li>wean only positives, sac negatives (mosaics?) </li></ul></ul><ul><ul><li>house male and females separately </li></ul></ul><ul><ul><li>mate at 6 weeks </li></ul></ul>
  39. 45. Housing <ul><li>Range from conventional to barrier </li></ul><ul><li>Researcher can usually advise on level of protection that is appropriate </li></ul>
  40. 46. Health Monitoring Programs <ul><li>Costly </li></ul><ul><li>Monitor health status of colony </li></ul><ul><li>Long-term savings: time, effort, money </li></ul><ul><li>Inform investigator (collaborators) of pathogen status </li></ul><ul><li>Prevent entry of pathogens </li></ul><ul><li>Promptly detect and deal/eliminate pathogen entry </li></ul>
  41. 47. Health Monitoring Programs <ul><li>Months of research data may have to be thrown out because of undetected infection </li></ul><ul><ul><li>Unfit for research </li></ul></ul><ul><ul><li>Data unreliable </li></ul></ul>
  42. 48. Pathogens <ul><li>Viral, bacterial, parasitic, and fungal </li></ul><ul><ul><li>Sometimes no overt signs </li></ul></ul><ul><ul><li>Many alter host physiology - host unsuitable for many experimental uses </li></ul></ul><ul><li>Cures can be bad too! </li></ul><ul><ul><li>Parasiticide - Ivermectin - immune system-modulating activity </li></ul></ul>
  43. 49. Pathogens (cont): Some common pathogens and their effects <ul><li>Sendai virus </li></ul><ul><ul><li>Mouse, rat, hamsters </li></ul></ul><ul><ul><li>One of the most important mouse pathogens </li></ul></ul><ul><ul><li>Transmission - contact, aerosol - very contagious </li></ul></ul><ul><ul><li>Clinical signs - generally asymptomatic; minor effects on reproduction and growth of pups </li></ul></ul>
  44. 50. Pathogens (cont): Some common pathogens and their effects <ul><ul><li>Infected shortly after birth </li></ul></ul><ul><ul><li>No carrier state - stop breeding </li></ul></ul><ul><ul><li>Altered physiology: as the virus travels down the resp.. tract -necrosis of airway epithelium, pneumonia in lungs, lesions. </li></ul></ul><ul><ul><li>129/J and DBA, aged and immunodeficient most susceptible; SJL/J and C57Bl/6 most resistant </li></ul></ul>
  45. 51. Pathogens (cont): Some common pathogens and their effects <ul><li>Reported effects </li></ul><ul><ul><li>Interference with early embryonic development and fetal growth </li></ul></ul><ul><ul><li>Alterations of macrophage, natural killer (NK) cell, and T- and B-cell function </li></ul></ul><ul><ul><li>Pulmonary hypersensitivity </li></ul></ul><ul><ul><li>Isograft rejection </li></ul></ul><ul><ul><li>Wound healing </li></ul></ul>
  46. 52. Pathogens (cont): Some common pathogens and their effects <ul><li>MHV </li></ul><ul><ul><li>Probably most important pathogen of laboratory mice </li></ul></ul><ul><ul><li>Extremely contagious; aerosol, direct contact; fomites </li></ul></ul><ul><ul><li>No carrier state </li></ul></ul><ul><ul><li>Clinic state: varies dependent upon MHV and mouse strains </li></ul></ul>
  47. 53. Pathogens (cont.): Some common pathogens and their effects <ul><ul><li>Diarrhea, poor growth, death </li></ul></ul><ul><ul><li>Immunodeficient (e.g. nu/nu) wasting syndrome -eventual death </li></ul></ul><ul><ul><li>Immunocompromised reported effects: necrotic changes in several organs, including liver, lungs, spleen, intestine, brain, lymph nodes, and bone marrow; differentiation of cells bearing T-lymphocyte markers; altered enzyme activities, bilirubin concentration, enhanced phagocytic activity of macrophages, rejection of xenograft tumors etc. etc. etc. </li></ul></ul>
  48. 54. Pathogens (cont.): Some common pathogens and their effects <ul><li>Helicobacter spp </li></ul><ul><ul><li>Genus keeps expanding with discoveries </li></ul></ul><ul><ul><li>H. Hepaticus (mice) most prominent </li></ul></ul><ul><ul><li>Transmission: direct fecal-oral or fomites </li></ul></ul><ul><ul><li>Clinical signs absent in immunocompetent </li></ul></ul><ul><ul><li>Immunodeficient - rectal prolapse </li></ul></ul><ul><ul><li>Pathological changes: chronic, active hepatitis, enterocolitis, hepatocellular neoplasms </li></ul></ul>
  49. 55. Pathogens (cont.): Some common pathogens and their effects <ul><ul><li>Reported effects: confounds carcinogenicity research; gastointestinal system research </li></ul></ul>
  50. 56. Pathogens (cont.): Some common pathogens and their effects <ul><li>Oxyuriasis (Pinworms) </li></ul><ul><ul><li>Mouse pinworms (Syphacia obvelata) has been reported to infect humans </li></ul></ul><ul><ul><li>Eggs excreted in faeces, can aerosolize - wide spread environmental contamination </li></ul></ul><ul><ul><li>Infection rate high; infection usually sub clinical </li></ul></ul><ul><ul><li>Athymic (nu/nu) mice are more susceptible </li></ul></ul>
  51. 57. Pathogens (cont.): Some common pathogens and their effects <ul><ul><li>Few reports documenting the effects of pinworms on research, many consider irrelevant </li></ul></ul><ul><li>Acariasis (mites) </li></ul><ul><ul><li>Hairless mice not susceptible </li></ul></ul><ul><ul><li>Transmission - direct contact </li></ul></ul><ul><ul><li>Eradication very labour-intensive </li></ul></ul>
  52. 58. Pathogens (cont.): Some common pathogens and their effects <ul><ul><li>C57Bl very susceptible </li></ul></ul><ul><ul><li>Infestation: asymptomatic or may cause wasting; scruffiness; pruritus; patchy alopecia; accumulation of fine bran-like material, mostly over affected areas; self-trauma to the point of amputation; and secondary pyoderma </li></ul></ul><ul><ul><li>Pathological changes: hyperkeratosis, erythema, mast cell infiltration, ulcerative dermatitis, splenic lymphoid and lymph node hyperplasia; </li></ul></ul>
  53. 59. Pathogens (cont.): Some common pathogens and their effects <ul><li>Reported to have caused: </li></ul><ul><ul><li>altered behaviour </li></ul></ul><ul><ul><li>selective increases in immunoglobulin G1 (IgG1), IgE, and IgA levels and depletion in IgM and IgG3 levels in serum </li></ul></ul><ul><ul><li>Lymphocytopenia </li></ul></ul><ul><ul><li>Granulocytosis </li></ul></ul><ul><ul><li>Increased production of IL-4; decreased production of IL-2 </li></ul></ul>
  54. 60. The End and Good bye!