1. Transgenic mice: generation and husbandry

2. Transgenic vs. “knock-out”
Transgenic: an organism that has had DNA introduced into one or more of its cells artificially
“transgenic”: DNA is integrated in a random fashion by injecting it into the pronucleus of a fertilized ovum
Random (approx.. 10% disrupt an endogenous gene important for normal development)
multiple copies

3. Transgenic vs. “knock-out”
Transgenic: an organism that has had DNA introduced into one or more of its cells artificially
“transgenic”: DNA is integrated in a random fashion by injecting it into the pronucleus of a fertilized ovum
Random (approx.. 10% disrupt an endogenous gene important for normal development)
multiple copies

4. Transgenic vs. “knock-out”
“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
targeted insertion

6. Transgenic production
Transgenic mice are often generated to
1. characterize the ability of a promoter to direct tissue-specific gene expression
e.g. a promoter can be attached to a reporter gene such as LacZ or GFP
2. examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

7. Brinster's growth hormone mouse

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)

9. GFP transgenic mouse (Nagy)
9.5 day embryos -
GFP and wt
Tail tip

10. GFP transgenic mouse (Nagy)

12. Planning a Transgenic production mouse colony
Mouse strain - popular
Colony size
typical injection 200 embryos (7-10 females s.o.)
Superovulation efficiency
Parenting suitability
Pseudo-pregs

14. Injecting fertilized eggs
The eggs are harvested 0.5 dpc (superovulated or natural matings)
The DNA is usually injected into the male pronucleus
The eggs can be transferred the same day or the next (2-cell) into pseudopregnant female oviducts

15. Pronuclear injection

16. Implantation of 1 or 2 cell embryos
The injected eggs are implanted the same day or are incubated overnight and implanted the next day
Injected eggs are transferred to the oviduct of a 0.5 dpc pseudopregnant female

17. Implanting 1(or 2) cell embryos

18. Implanting 1(or 2) cell embryos (cont.)3

19. Pseudopregnant females and vasectomized males
Female mice can be tricked into thinking they are pregnant
A mouse in estrus is mated with a vasectomized male
pseudopregnancy
If eggs (blastocysts) implanted will become truly pregnant and will give birth to live offspring

20. Vasectomizing 1 2

21. Breeding Tg founders Individually backcrossed to the strain of choice
DO NOT intercross different founders - each founder results from a separate RANDOM transgene integration even

22. Transgenic mice as tools
Study gene function
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
Drug testing

23. Transgenic mice as tools
Polio virus receptor
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.
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

26. Vector design Recombinant DNA methods: Simple KO
Structural gene desired (e.g. insulin gene) to be "knocked out" is replaced partly or completely by a positive selection marker. (knock out function!)
Vector DNA to enable the molecules to be inserted into host DNA molecules

27. Typical KO vector
*tk:thymidine kinase

28. Embryonic stem cells
Harvested from the inner cell mass of mouse blastocysts
Grown in culture and retain their full potential to produce all the cells of the mature animal, including its gametes

29. ES cells growing in culture

30. ES cells are transformed
Cultured ES cells are exposed to the vector
Electroporation punched holes in the walls of the ES cells
Vector in solution flows into the ES cells
The cells that don't die are selected for transformation using the positive selection marker
Randomly inserted vectors will be killed by gancyclovir

32. Successfully transformed ES cells are injected into blastocysts

33. Implantation of blastocysts
The blastocysts are left to rest for a couple of hours
Expanded blastocysts are transferred to the uterine horn of a 2.5 dpc pseudopregnant female
Max. 1/3 of transferred blasts will develop into healthy pups

34. Implanting blastocysts1 2

35. Implanting blastocysts (cont.)3 4

36. Littermates Black mouse - no apparent ES cell contribution
Chimeric founder -
strong ES cell
contribution
Chimeric founder -
weaker ES cell
contribution

37. Chimeric mouse

38. Testing the offspring
A small piece of tissue - tail or ear - is examined for the desired gene
10-20% will have it and they will be heterozygous for the gene

39. Breeding Chimeras (knock-out founder)
Chimera - the founder
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.

40. Breeding Chimeras (knock-out founder)cont
Males that are 40% to 100% based on agouti coat colour should be bred
Females should not be bred (low incidence of success) ES cells are male.
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

41. Knock-out mice as tools
If the replacement gene is nonfunctional (null allele), mating of the heterozygous will produce a strain of "knock-outs' homozygous for the nonfunctional gene (both copies are knocked-out
Find out if the gene is indispensable (suprisingly many are not!)
"pleiotropic" expression in different tissues in different ways and at different times in development

42. Breeding Transgenics
Most transgenics are bred onto a C57Bl/6 background
standard
BL/6 breeding information
mate 6-8 weeks for best reproductive performance
replace males when 1 year old

43. Breeding Transgenics (cont.)
Replace females after 6 litters or at 6 months of age
quick breeding - 1 founder male: 2 females
rotation of females through male cage
Common problems:
female not good mother, check for milk - give auntie
male cannibalizing litter
fighting (separate) Do not “reunite” males

44. Breeding Transgenics (cont)
Stick to schedules or be overwhelmed
strict records (birth, ID, parents)
ID pups
tail tip or collect ear tissue at 2 weeks
try to genotype before weaning
wean only positives, sac negatives (mosaics?)
house male and females separately
mate at 6 weeks

45. Housing Range from conventional to barrier
Researcher can usually advise on level of protection that is appropriate

46. Health Monitoring Programs
Costly
Monitor health status of colony
Long-term savings: time, effort, money
Inform investigator (collaborators) of pathogen status
Prevent entry of pathogens
Promptly detect and deal/eliminate pathogen entry

47. Health Monitoring Programs
Months of research data may have to be thrown out because of undetected infection
Unfit for research
Data unreliable

48. Pathogens Viral, bacterial, parasitic, and fungal
Sometimes no overt signs
Many alter host physiology - host unsuitable for many experimental uses
Cures can be bad too!
Parasiticide - Ivermectin - immune system-modulating activity

49. Pathogens (cont): Some common pathogens and their effects
Sendai virus
Mouse, rat, hamsters
One of the most important mouse pathogens
Transmission - contact, aerosol - very contagious
Clinical signs - generally asymptomatic; minor effects on reproduction and growth of pups

50. Pathogens (cont): Some common pathogens and their effects
Infected shortly after birth
No carrier state - stop breeding
Altered physiology: as the virus travels down the resp.. tract -necrosis of airway epithelium, pneumonia in lungs, lesions.
129/J and DBA, aged and immunodeficient most susceptible; SJL/J and C57Bl/6 most resistant

51. Pathogens (cont): Some common pathogens and their effects
Reported effects
Interference with early embryonic development and fetal growth
Alterations of macrophage, natural killer (NK) cell, and T- and B-cell function
Pulmonary hypersensitivity
Isograft rejection
Wound healing

52. Pathogens (cont): Some common pathogens and their effects
MHV
Probably most important pathogen of laboratory mice
Extremely contagious; aerosol, direct contact; fomites
No carrier state
Clinic state: varies dependent upon MHV and mouse strains

53. Pathogens (cont.): Some common pathogens and their effects
Diarrhea, poor growth, death
Immunodeficient (e.g. nu/nu) wasting syndrome -eventual death
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.

54. Pathogens (cont.): Some common pathogens and their effects
Helicobacter spp
Genus keeps expanding with discoveries
H. Hepaticus (mice) most prominent
Transmission: direct fecal-oral or fomites
Clinical signs absent in immunocompetent
Immunodeficient - rectal prolapse
Pathological changes: chronic, active hepatitis, enterocolitis, hepatocellular neoplasms

55. Pathogens (cont.): Some common pathogens and their effects
Reported effects: confounds carcinogenicity research; gastointestinal system research

56. Pathogens (cont.): Some common pathogens and their effects
Oxyuriasis (Pinworms)
Mouse pinworms (Syphacia obvelata) has been reported to infect humans
Eggs excreted in faeces, can aerosolize - wide spread environmental contamination
Infection rate high; infection usually sub clinical
Athymic (nu/nu) mice are more susceptible

57. Pathogens (cont.): Some common pathogens and their effects
Few reports documenting the effects of pinworms on research, many consider irrelevant
Acariasis (mites)
Hairless mice not susceptible
Transmission - direct contact
Eradication very labour-intensive

58. Pathogens (cont.): Some common pathogens and their effects
C57Bl very susceptible
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
Pathological changes: hyperkeratosis, erythema, mast cell infiltration, ulcerative dermatitis, splenic lymphoid and lymph node hyperplasia;

59. Pathogens (cont.): Some common pathogens and their effects
Reported to have caused:
altered behaviour
selective increases in immunoglobulin G1 (IgG1), IgE, and IgA levels and depletion in IgM and IgG3 levels in serum
Lymphocytopenia
Granulocytosis
Increased production of IL-4; decreased production of IL-2

60. The End and Good bye!