1. Inject each animal intraperitoneally (see Note 5) with 1 ml of
the input pool with an insulin syringe U-100 with 26 G Â ½
00
needle (see Note 6).
2. Monitor animals daily until they are terminated at a predetermined date. Weigh animals and look for endpoint criteria,
which include loss of appetite, ruffled fur, abnormal gait, prostration, and loss of more than 10% of the animal’s maximum
weight.
3. Euthanize animals by isoflurane inhalation (4–5%) in an induction chamber followed by thoracotomy.
4. Collect blood by cardiac puncture with a 3 ml syringe with a
25 G Â 5/8
00 needle. Immediately transfer blood into an
EDTA blood collection tube, and mix well by inversion (five
to six times) (see Note 7).
5. Harvest tissues and store them in cryotubes at À80
C for use
in step 1 of Subheading 3.4 (see Note 8).
3.3 Fitness Testing
In Vitro
1. Collect 10 ml of the input pool, and spin it down for 20 min at
3200 Â g in a 50 ml centrifuge tube. Store the pellet at À80
C
until use in step 4 of Subheading 3.4.
2. Inoculate three 125 ml screw-capped Erlenmeyer flasks containing 25 ml EMJH with 1 ml of the input pool. Incubate the
cultures under the desired conditions (see Note 9).
3. After a predetermined time or when cultures reach density % 1 Â 10
8 /ml, spin down each culture for 20 min at
3220 Â g in 50 ml centrifuge tubes (see Note 10).
4. Store cell pellets at À80
C for use in step 2 of Subheading 3.4.
3.4 DNA Extraction
1. For in vitro cultures, skip to step 2. To prepare animal tissues
for DNA extraction, dice 50–80 mg of tissue, and transfer it
into a 1.5 ml microcentrifuge tube with 500 μl PBS. Homogenize tissue with the Omni Bead Ruptor for 1 min at five movements per second. Determine the volume corresponding to
25 mg of tissue (see Note 11), and transfer it into a 1.5 ml
microcentrifuge tube.
2. Extract the DNA with a DNA extraction kit following the
manufacturer’s instructions. Elute DNA with 100 μl elution
buffer.
3. For blood, extract DNA from 100 μl blood collected in an
EDTA blood collection tube using a DNA extraction kit.
Elute the DNA with 100 μl elution buffer.
4. For the input pool, extract DNA from pellet using a DNA
extraction kit following the manufacturer’s instructions. Elute
the DNA with 100 μl elution buffer.
Transposon Sequencing in Leptospira interrogans
101
the input pool with an insulin syringe U-100 with 26 G Â ½
00
needle (see Note 6).
2. Monitor animals daily until they are terminated at a predetermined date. Weigh animals and look for endpoint criteria,
which include loss of appetite, ruffled fur, abnormal gait, prostration, and loss of more than 10% of the animal’s maximum
weight.
3. Euthanize animals by isoflurane inhalation (4–5%) in an induction chamber followed by thoracotomy.
4. Collect blood by cardiac puncture with a 3 ml syringe with a
25 G Â 5/8
00 needle. Immediately transfer blood into an
EDTA blood collection tube, and mix well by inversion (five
to six times) (see Note 7).
5. Harvest tissues and store them in cryotubes at À80
C for use
in step 1 of Subheading 3.4 (see Note 8).
3.3 Fitness Testing
In Vitro
1. Collect 10 ml of the input pool, and spin it down for 20 min at
3200 Â g in a 50 ml centrifuge tube. Store the pellet at À80
C
until use in step 4 of Subheading 3.4.
2. Inoculate three 125 ml screw-capped Erlenmeyer flasks containing 25 ml EMJH with 1 ml of the input pool. Incubate the
cultures under the desired conditions (see Note 9).
3. After a predetermined time or when cultures reach density % 1 Â 10
8 /ml, spin down each culture for 20 min at
3220 Â g in 50 ml centrifuge tubes (see Note 10).
4. Store cell pellets at À80
C for use in step 2 of Subheading 3.4.
3.4 DNA Extraction
1. For in vitro cultures, skip to step 2. To prepare animal tissues
for DNA extraction, dice 50–80 mg of tissue, and transfer it
into a 1.5 ml microcentrifuge tube with 500 μl PBS. Homogenize tissue with the Omni Bead Ruptor for 1 min at five movements per second. Determine the volume corresponding to
25 mg of tissue (see Note 11), and transfer it into a 1.5 ml
microcentrifuge tube.
2. Extract the DNA with a DNA extraction kit following the
manufacturer’s instructions. Elute DNA with 100 μl elution
buffer.
3. For blood, extract DNA from 100 μl blood collected in an
EDTA blood collection tube using a DNA extraction kit.
Elute the DNA with 100 μl elution buffer.
4. For the input pool, extract DNA from pellet using a DNA
extraction kit following the manufacturer’s instructions. Elute
the DNA with 100 μl elution buffer.
Transposon Sequencing in Leptospira interrogans
101