2.2.5 Mouse Virulence
1. 0.85% NaCl.
2. 5% mucin.
2.2.6 Mouse
Protection Test
1. 0.2 ml of a bacterial suspension containing 10
7 live Ty 21a/ml.
2. 0.2 ml of a suspension containing 10
8 viable Ty 21a cells.
2.3 Viral Attenuation
by Sequential
Passaging [13]
2.3.1 Vaccine
Preparation
1. Oka strain varicella virus.
2. Human embryonic lung (HEL) cells and GPE cells.
3. Human diploid cells (WI-38) cells (Flow Laboratories).
4. Roux bottles.
5. Skin and muscle tissues from 3 to 4-week-old guinea pig
embryos.
6. Phosphate-buffered saline solution (PBS): 8 g NaCl, 0.2 g KCl,
1.44 g NaHPO 4 , and 0.24 g KH 2 PO 4 dissolved in 800 ml. pH
is adjusted to 7.4. Volume is made up to 1 L (see Note 1).
7. Edetic acid treatment.
8. Eagle’s minimum essential medium.
9. 20KC sonifier.
10. Centrifuge.
11. 5% sugar.
2.4 MicroRNA-based
Viral Attenuation [14]
2.4.1 Eggs and Cell
Culture
1. Embryonated chicken eggs purchased from Charles River
Laboratories, CT. MDCK cells (ATCC, #CCL-34), MEM
(Sigma).
2. 10% FBS (Gibco, NY).
3. 50 μg/ml gentamicin.
4. 1 mM sodium pyruvate.
5. HEK293T (ATCC, #CRL-11268), MEF (ATCC, #CRL2214), MEF DicerÀ/À (provided by Dr. Wu).
6. DF1 (ATCC, #CRL-12203).
7. A549 (ATCC, #CCL-185) cells.
8. DMEM (Gibco, NY).
9. 1% penicillin.
10. 1 μg/ml streptomycin (Gibco, NY).
2.4.2 Artificial microRNA
Design and Expression
1. miR-93 cassette with a scrambled control sequence, the
miR-93 locus, and amiR-93NP synthesized by GenScript.
2. microRNA-expressing plasmid pLL3.7.
3. 293T cells.
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1. 0.85% NaCl.
2. 5% mucin.
2.2.6 Mouse
Protection Test
1. 0.2 ml of a bacterial suspension containing 10
7 live Ty 21a/ml.
2. 0.2 ml of a suspension containing 10
8 viable Ty 21a cells.
2.3 Viral Attenuation
by Sequential
Passaging [13]
2.3.1 Vaccine
Preparation
1. Oka strain varicella virus.
2. Human embryonic lung (HEL) cells and GPE cells.
3. Human diploid cells (WI-38) cells (Flow Laboratories).
4. Roux bottles.
5. Skin and muscle tissues from 3 to 4-week-old guinea pig
embryos.
6. Phosphate-buffered saline solution (PBS): 8 g NaCl, 0.2 g KCl,
1.44 g NaHPO 4 , and 0.24 g KH 2 PO 4 dissolved in 800 ml. pH
is adjusted to 7.4. Volume is made up to 1 L (see Note 1).
7. Edetic acid treatment.
8. Eagle’s minimum essential medium.
9. 20KC sonifier.
10. Centrifuge.
11. 5% sugar.
2.4 MicroRNA-based
Viral Attenuation [14]
2.4.1 Eggs and Cell
Culture
1. Embryonated chicken eggs purchased from Charles River
Laboratories, CT. MDCK cells (ATCC, #CCL-34), MEM
(Sigma).
2. 10% FBS (Gibco, NY).
3. 50 μg/ml gentamicin.
4. 1 mM sodium pyruvate.
5. HEK293T (ATCC, #CRL-11268), MEF (ATCC, #CRL2214), MEF DicerÀ/À (provided by Dr. Wu).
6. DF1 (ATCC, #CRL-12203).
7. A549 (ATCC, #CCL-185) cells.
8. DMEM (Gibco, NY).
9. 1% penicillin.
10. 1 μg/ml streptomycin (Gibco, NY).
2.4.2 Artificial microRNA
Design and Expression
1. miR-93 cassette with a scrambled control sequence, the
miR-93 locus, and amiR-93NP synthesized by GenScript.
2. microRNA-expressing plasmid pLL3.7.
3. 293T cells.
334
Dipasree Hajra et al.