Development of Transgenic Mice

1
Development of Transgenic Mice
2
Development of lefty transgenic mouse
Non-regulated, Non-tissue specific, Transgene
expression
Promoter
Transgene
3
Development of lefty transgenic mouse
Bigenic regulation of transgene expression
Non-regulated, Tissue specific, Transgene
expression
Transcription factor
Responsive Promoter
Transgene
Constitutive, transgene expression
4
Development of lefty transgenic mouse
Bigenic regulation of transgene expression
Regulated, Tissue specific, Transgene expression
Tissue specific Promoter
Transcription factor
Tetracycline Ecdysone RU486
Ligand
Transcription factor
Transgene
Responsive Promoter
Gene switch, transgene expression
5
Development of lefty transgenic mouse
tet off tet on regulation of transgene
expression
Regulated, Tissue specific, Transgene expression
Tissue specific Promoter
Transcription factor
Modified Chimeric Tet-R tTA (activation domain
of Herpes Simplex Virus thymidine kinase HSV
VP-16)
Transcription factor
ON
Transgene
Promoter (tet-O)
Gene switch, transgene expression
6
Development of lefty transgenic mouse
tet off tet on of transgene expression
Regulated, Tissue specific, Transgene expression
Tissue specific Promoter
Transcription factor
Modified Chimeric Tet-R tTA (activation domain
of Herpes Simplex Virus thymidine kinase HSV
VP-16)
Ligand
Tetracycline
Transcription factor
OFF
Transgene
Promoter (tet-O)
Gene switch, transgene expression
7
Development of lefty transgenic mouse
Mifepristone (RU-486) gene switch
Regulated, Tissue specific, Transgene expression
Tissue specific Promoter
Transcription factor
Modified human PR (GL-VP) Mutant LBD does not
bind P but binds RU-486. DBD of yeast
transcription factor, gal14 Transactivation
domain of HSV VP16
Transcription factor
OFF
Transgene
Promoter (UASG)
Gene switch, transgene expression
8
Development of lefty transgenic mouse
Mifepristone (RU-486) gene switch
Regulated, Tissue specific, Transgene expression
Tissue specific Promoter
Transcription factor
Modified human PR (GL-VP) Mutant LBD does not
bind P but binds RU-486. DBD of yeast
transcription factor, gal14 Transactivation
domain of HSV VP16
Ligand
RU-486
Transcription factor
ON
Transgene
Promoter (UASG)
Gene switch, transgene expression
9
Development of lefty transgenic mouse
Development
Identifcation Propagation Maintenence
10
Development of lefty transgenic mouse
Timeline for transgenic mouse development
Superovulation
Mating
Birth
DNA and Embryo transfer
5 IU of human chorionic gonadotropin (hCG)
Mating with stud males
5 IU of pregnant mare serum gonadotropin (PMSG)
Sacrifice
Examination of newborns
DNA micro-injection
Embryo Transfer to pesudo-pregnant females
-1
Day
-3
18-21
0
1230-1400
1300-1600
1400-1700
1300-1600
1200-1400
1300
Hour
Light cycle
? Removal of oviduct and placement in 2 ml M16
medium supplemented with hyaluronidase ?
Isolation of the distented area containing the
fertilized eggs ? Extrusion of eggs into the
medium
Light on 8 AM Light off 10 PM
11
Development of lefty transgenic mouse
Timeline for transgenic mouse analysis

• ? DNA preparation and purification
• ? Production of superovulated female donors
• ? Mating of the superovulated female donors with
  males
• ? Retrieval of fertilized eggs from female donors
  for micro-injection
• ? Micro-injection of eggs with DNA
• ? Production of F1 foster pseudo-pregnant females
• ? Production of sterile male studs (vasectomy)
• ? Mating of pseudo-pregnant females with
  vasectomized males
• ? Transfer of injected eggs to pseudo-pregnant
  females
• ? Birth of pups
• ? Tagging of the pups
• ? Tail biopsy and DNA analysis
• ? Establishing lines
• ? Embryo freezing

12
Development of lefty transgenic mouse
Timeline for transgenic mouse analysis

• ? DNA preparation and purification
• ? Production of superovulated female donors
• ? Mating of the superovulated female donors with
  males
• ? Retrieval of fertilized eggs from female donors
  for micro-injection
• ? Micro-injection of eggs with DNA
• ? Production of F1 foster pseudo-pregnant females
• ? Production of sterile male studs (vasectomy)
• ? Mating of pseudo-pregnant females with
  vasectomized males
• ? Transfer of injected eggs to pseudo-pregnant
  females
• ? Birth of pups
• ? Tagging of the pups
• ? Tail biopsy and DNA analysis
• ? Establishing lines
• Embryo freezing
• Microinjection of DNA under a differential
  contact microscope using siliconized glass
  capillary (1 mm OD, 0.78 mm ID, Cat No 30-30-0,
  Frederick Haer Co, Brunswick, ME)

13
Development of lefty transgenic mouse
DNA preparation and purification ? Preparation
of linearized and double digested construct
(Digestion of 30 micrograms of DNA by restriction
enzymes) ? Extraction twice with phenol/chlorform
? Precipitation with acetate/ethanol ?
Separation of the digested DNA by zonal sucrose
gradient centrifugation ? Collection of
fractions ? Electrophoresis of the aliquoted
fractions to identify fraction containing
insert ? Pooling and concentration of the
fractions in Centricon 100
14
Development of lefty transgenic mouse

• Production of superovulated female donors
• Superovulation
• ? Light cycle from 0630 to 1830 hr (light on at 8
  Am, light off 10 PM)
• ? Injection of fertilized eggs ( 35 1-cell stage
  eggs, two to three batches) at 1400 hr (6 hr
  before cleavage)
• ? Injection of fertilized eggs ( 35 2-cell stage
  eggs cultured overnight in M2 medium at 37 C in
  5 CO2, two to three batches, (after the first
  cleavage)
• ? Predict successful pregnancy rate 80
  non-injected eggs. 30 injected eggs
• Maintain 50, 30 gram, mature CD1 female mice for
  superovulation.
• Housed in groups of 10 to avoid synchronization
  of estrus cycles.
• Animals within one cage might become synchronized
  but the first estrous day remains different among
  cages

15
Development of lefty transgenic mouse

• Production of superovulated female donors
• Superovulation
• Induction of superovulation by intraperitoneal
  injection of F1, 3-5 week old, female mice or
  mature 8 week old females with
• ? Day -3 5 IU of pregnant mare serum
  gonadotropin (PMSG) 1300 hr
• Day -1 5 IU of hCG 48 hours later 1200-1400 hr.
  Mating with males studs
• ? Day 0 Sacrifice 12300-1400 hr
• ? Removal of oviduct and placement in 2 ml M16
  medium supplemented with hyaluronidase
• ? Isolation of the distented area containing the
  fertilized eggs
• ? Extrusion of eggs into the medium

16
Development of lefty transgenic mouse
Micro-injection of DNA into pro-nucleus of
fertilized eggs ? DNA concentration 2.5
microgram/ml in 10 mM Tris-HCl, pH 7.5, 0.1 mM
EDTA, stored at 20 C ? Centrifuge an aliquot at
13,000 rpm in a microcentrifuge before use to
remove particulates ? Inject using the Eppendorf
gas injector (Eppendorf microinjector 5242, Carl
Zeiss, Germany) with injection pressure of 100
psi and back pressure set at 1/3 of injection
pressure ? Frequency of DNA integration at least
25
17
Development of lefty transgenic mouse

• Production of sterile male studs
• Vasectomy of 6 week old mice
• ? Anesthesia
• ? Ventral incision
• ? Identification of vas deferens
• Cauterization of the vas deferens
• Maintain 30 males as breeders

18
Development of lefty transgenic mouse

• Transfer of injected eggs to pseudo-pregnant
  foster females
• Foster mothers are
• ? mated without superovulation
• ? mated with vasectomized male (mating of female
  mice with stud males used not more than twice a
  week and not more than 6 months of age (frequency
  of mating 75) 1300-1600 hr
• ? kept in five groups of 5 to increase chance of
  mating on each day
• ? change foster mothers and vasectomized mice
  every 6-8 months

19
Development of lefty transgenic mouse
Tail biopsy and DNA analysis ? Removal of 1/3 to
1/2 of tail of 3-5 week old mice ? Cutting the
tail into 5-10 pieces ? Placement of tail pieces
in 500 ml 10 mM NaCl, 10 mM Tris HCl, pH 8, 10 mM
EDTA, pH 8, 2 SDS ? Addition of proteinase K (20
mg/ml) ? Incubation at 37 C on a rocking shaker,
2-2.5 hr
20
Development of lefty transgenic mouse
Tail biopsy and DNA analysis ? Addition of 300 ml
DNA grade phenol ? Extraction of DNA with 150 ml
phenol ? Extraction with 100 ml
chloroform-isoamyl alcohol (241 v/v) ?
Precipitation of DNA with 2.5 volumes of ice-cold
100 ethanol ? Dissolving DNA pellet in 100-200
ml of 10 mM Tris HCl pH 8, 0.1 mM EDTA pH 8
(final concentration of DNA, 2-3 mg/ml)
21
Development of lefty transgenic mouse
Establishing lines and embryo freezing for
constitution of the strain ? Identifying the
positive progeny ? Establishing homozygous line ?
Confirming the identity of the homozygous line by
Southern Blotting ? Breeding aggressively until
line is established ? Large numbers of embryos
required to assure rapid constitution of the
strain
22
Development of lefty transgenic mouse
Reasons for failure ? DNA co-transfer
(Insufficient purity of DNA from plasmid) ? Lack
of enhancer elements ? Lack of trans-acting
elements ? Delayed integration of DNA
(mosaicism) ? Unstable concatamers-Structural
alterations Deletion Recombination Duplication
Translocation ? Dilution of DNA ? Improper
injection ? Lethality of embryos