Transgenic mice: generation and husbandry

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 2 1

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 blastocysts 1 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