1. Transgenic animals and knockout animals

2. 3 main ways to do biological research:
Do research in test tubes.
Do research with cells.
Do research directly with animals.

3. Transgenic animals and knockout animals
Part 1: Transgenic animals:
Introduction to transgenic animals.
How to make transgenic animals?
How to make conditional transgenic animals?
Applications of transgenic animals.
Part 2: Knockout animals
Introduction to knockout animals.
How to make knockout animals?
How to make conditional knockout animals?
Applications of knockout animals.

4. Transgenic Animal
Animal has one or more foreign genes inserted into chromosome DNA inside its cells artificially.
After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst, foreign gene is inserted in a random fashion into chromosome DNA:
Randomly (Foreign gene may disrupt an endogenous gene important for normal development, and the chance is about 10%. )
multiple copies

5. Transgenic animals and knockout animals
Part 1: transgenic animals:
Introduction to transgenic animals.
How to make transgenic animals?
How to make conditional transgenic animal?
Applications of transgenic animals.
Part 2: Knockout animals
Introduction to knockout animals.
How to make knockout animals?
How to make conditional knockout animals?
Applications of knockout animal.

6. ES cell transformation
Injection of gene into fertilized egg

7. Method 1: ES cell transformation vs
Method 1: ES cell transformation vs. Method 2: Injection of gene into fertilized egg
1. ES cell transformation works well in mice only.
Other transgenic animals are produced by egg injection
2. ES cell transformation provides more control of the integration step
(selection of stably transfected ES cells)
3. Injection of gene into fertilized egg is less reliable
(viability of eggs, frequency of integration),
but it helps to avoids chimeric animals

9. Injecting fertilized eggs
The eggs are harvested from mice (superovulated or natural matings).
The DNA is usually injected into the male pronucleus.
The eggs can be transferred in the same day (1 cell) or the next day (2-cells) into pseudopregnant female oviducts.

11. Breeding Transgenic animals (transgenic founders)
Transgenic animals Individually are backcrossed to non-transgenic animals.
DO NOT intercross different founders. Each founder results from a separate RANDOM transgene integration event.

12. Transgenic animals and knockout animals
Part 1: transgenic animals:
Introduction to transgenic animals.
How to make transgenic animals?
How to make conditional transgenic animals?
Applications of transgenic animals.
Part 2: Knockout animals
Introduction to knockout animals.
How to make knockout animals?
How to make conditional knockout animals?
Applications of knockout animal.

13. Conditional Transgenic mouse
The expression of transgene in transgenic mouse can be induced

14. Important Considerations for Conditional Transgenes
Transgenes have low or no expression when not induced
Large difference between induced and non-induced gene expression
Transgene expression rapidly turns on or off.
Inducer (doxycycline, tamoxifen, cre) is not toxic and easily administered

15. Tetracycline Controlled Transactivator tTA “Tet-off”
VP16
Doxycycline blocks tTA DNA binding
tTA binds to tetO to activate transcription

16. Reverse Tetracycline Controlled Transactivator tTA “Tet-on”
rtetR
VP16
Doxycycline allows rtTA to bind to tetO
Without doxcycline rtTA can not bind to tetO

17. Tetracycline Regulation: Summary
No Doxycycline Doxycycline
tTA expressed not expressed
rtTA not expressed expressed

18. Transgenic animals and knockout animals
Part 1: transgenic animals:
Introduction to transgenic animals.
How to make transgenic animals?
How to make conditional transgenic animal?
Applications of transgenic animals.
Part 2: Knockout animals
Introduction to knockout animals.
How to make knockout animals?
How to make conditional knockout animals?
Applications of knockout animal.

19. Applications of Transgenic Animals
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
3 Study gene function
Many human diseases can be modeled by introducing the same mutation into the mouse. Intact animal provides a more complete and physiologically relevant picture of a transgene's function than in vitro testing.
4. Drug testing

20. Example 1: Transgenic Cattle
Cloned transgenic cattle produce milk with higher levels of beta-caein and k-casein
Published in Nature, Jan, 2003

23. Example 2: Transgenic Mouse
The growth hormone gene has been engineered to be expressed
at high levels in animals.
The result: BIG ANIMALS
Mice fed with heavy metals are 2-3 times larger
Metallothionein
promoter
regulated by heavy metals

24. Example 3: Transgenic Mouse
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.

25. Example 4: GFP transgenic mouse (Nagy)
9.5 day embryos -
GFP and wt
Tail tip

26. GFP transgenic mouse (Nagy)

27. Example 5: Wild and domestic trout respond differently
to overproduction of growth hormone.
So, GH is not effective to domestic trout.

28. Example 6: Transgenic mice as tools
Normal mice can't be infected with polio virus. They lack the cell-surface Polio virus receptor. But, human has Polio virus receptor.
Transgenic mice expressing the human gene for the Polio receptor can be infected by polio virus and even develop paralysis and other pathological changes characteristic of the disease in humans

29. Transgenic animals and knockout animals
Part 1: transgenic animals:
Introduction to transgenic animals.
How to make transgenic animals?
How to make conditional transgenic animal?
Applications of transgenic animals.
Part 2: Knockout animals
Introduction to knockout animals.
How to make knockout animals?
How to make conditional knockout animals?
Applications of knockout animals.

30. knock-out Animal
One endogenous gene in an animal is changed. The gene can not be expressed and loses its functions.
DNA is introduced first into embryonic stem (ES) cells.
ES cells that have undergone homologous recombination are identified.
ES cells are injected into a 4 day old mouse embryo: a blastocyst.
Knockout animal is derived from the blastocyst.

31. Transgenic animals and knockout animals
Part 1: transgenic animals:
Introduction to transgenic animals.
How to make transgenic animals?
How to make conditional transgenic animal?
Applications of transgenic animals.
Part 2: Knockout animals
Introduction to knockout animals.
How to make knockout animals?
How to make conditional knockout animals?
Applications of knockout animals.

33. 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 to knock out the gene functions.
Vector DNA to enable the molecules to be inserted into host DNA molecules

34. KNOCKOUT MICE Isolate gene X and insert it into vector.
Inactivate the gene
by inserting a marker gene
that make cell resistant
to antibiotic (e.g. Neomycin)
Normal (+) gene X
Genome
Defective (-)
Gene X
Transfer vector
with (-) gene X
into ES cells
(embryonic stem cells)
VECTOR
MARKER GENE
e.g.(NeoR)

35. Vector and genome will recombine via homologous sequences
Genomic gene
Exon 1
Exon 2
Exon 3
Exon 4
Homologous recombination
and gene disrution
Grow ES cells in
antibiotic containing media;
Only cell with marker gene
(without normal target gene)
will survive

36. Problems with homologous recombination
Unwanted random non-homologous recombination
is very frequent.
This method provides no selection against it
Solution: Replacement vectors
The knock-out construct contains the 1) NeoR gene
flanked by 2) two segments of the target gene
and 3) the HSVtk gene
Part of the gene replaced with NeoR
ES cells are selected for integration of NeoR and
against integration of HSVtk* (NeoR+/ HSVtk-) on gancyclovir

37. Replacement vectors NeoR+/ HSVtk- NeoR+/ HSVtk+ NeoR HSVtk Homologous
Gene segment 1
Gene segment 2
NeoR
HSVtk
Linearized replacement plasmid
NeoR
Homologous
recombination
NeoR+/ HSVtk-
Random integration
NeoR+/ HSVtk+
HSVtk will convert gancyclovir into a toxic drug and kill HSVtk+ cells

38. Typical KO vector
*tk:thymidine kinase

39. Inject ES cells with (-) gene X into early mouse embryo
Transfer embryos
to surrogate mothers
Resulting chimaras
have some cells
with (+) gene X
and (-) gene X.
Mate them with normal mice
It is lucky,
if germline contain (-) gene X
Screen pups to find -/+ and mate them
Next generation will split as 3:1
(Mendelian)

40. 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.

41. ES cells growing in culture

42. 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

43. Successfully transformed ES cells are injected into blastocysts

44. Implantation of blastocysts
The blastocysts injected with transformed ES cells are left to rest for a couple of hours
Expanded blastocysts are transferred to the uterine horn of a pseudopregnant female
Max. 1/3 of transferred blastocysts will develop into healthy pups

45. Implanting blastocysts1 2

46. Implanting blastocysts3 4

47. 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

48. Breeding Chimeras (knock-out founder)
Chimera - the founder
germ-line transmission - usually the ES cells are derived from a 129 mouse strain (agouti or white colour) and the ES cells are injected into blastocyst derived from a C57Bl/6 mouse (black).
The more that the ES cells contribute to the genome of the knockout mouse, the more the coat colour will be agouti. The chimera mouse is usually “tiger” striped.

49. Breeding Chimeras (knock-out founder)
Males that are 40% to 100% based on agouti coat colour should be bred
Females should not be bred (low incidence of success).
Breed aggressively- rotate females through male's cage. If the male produces more than 6 litters without transmitting knockout gene, the knockout gene will not likely go to germline and should not be used for more breeding.

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

51. Chimeric mouse

52. Transgenic animals and knockout animals
Part 1: transgenic animals:
Introduction to transgenic animals.
How to make transgenic animals?
How to make conditional transgenic animal?
Applications of transgenic animals.
Part 2: Knockout animals
Introduction to knockout animals.
How to make knockout animals?
How to make conditional knockout animals?
Applications of knockout animal.

53. Conditional knock-out animals How to make FLOXed gene
Gene of interest
NeoR TK
Electroporate targeting vector
into ES cells, followed
by +/- selection
loxP
loxP
NeoR+/ HSVtk-
cells selected
loxP
loxP
Gene flanked by loxP sites (floxed)
Make mice and breed floxed allele to homozygousity.

54. Mate FLOXed mice with mice carrying a Cre transgene
Marker gene
Promoter elements Cre IRES GFP SV40 p(A)
intron
Crucial element. Recombinase
would be expressed in accordance
with specificity of your promoter.
Promoter could be regulated !!!
artificailly or naturally

55. Conditional knock-out animals
inactivate a gene only in specific tissues
and at certain times during development and life.
Your gene of interest
is flanked by 34 bp loxP sites (floxed).
If CRE recombinase expressed
Gene between loxP sites is removed

56. Transgenic animals and knockout animals
Part 1: transgenic animals:
Introduction to transgenic animals.
How to make transgenic animals?
How to make conditional transgenic animal?
Applications of transgenic animals.
Part 2: Knockout animals
Introduction to knockout animals.
How to make knockout animals?
How to make conditional knockout animals?
Applications of knockout animal.

57. Applications of Knock-out animals
Find out if the gene is indispensable (suprisingly many are not!)
Check the phenotypes of knockout animals
Determine the functions of knockout gene.

58. 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

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

60. 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!

61. Pathogens: 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

62. Pathogens (cont): Some common pathogens and their effects
Infected shortly after birth
stop breeding
Altered physiology: as the virus travels down the respiratory tract -necrosis of airway epithelium, pneumonia in lungs, lesions.
129/J and DBA, aged and immunodeficient mice most susceptible; SJL/J and C57Bl/6 most resistant

63. 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
Wound healing

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

65. Pathogens (cont.): Some common pathogens and their effects
Diarrhea, poor growth, death
Immunodeficient (e.g. nu/nu) wasting syndrome -eventual death
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, enhanced phagocytic activity of macrophages, rejection of xenograft tumors etc.

66. Pathogens (cont.): Some common pathogens and their effects
Helicobacter spp
H. Hepaticus (mice) most prominent
Transmission: direct fecal-oral
Clinical signs absent in immunocompetent mice
In immunodeficient mice- rectal prolapse
Pathological changes: chronic, active hepatitis, enterocolitis, hepatocellular neoplasms

67. 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

68. 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

69. 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

70. The End and Good bye!