134
You, and Hunter [11] described a fluorescence resonance energy
transfer (FRET)-based biosensor for monitoring ATM kinase activity in live cells. Although this reporter provides direct measurement of the ATM kinase activity, it has a limitation of usability in
mouse model due to tissue penetration and autofluorescence in the
CFP-YFP range. In this book chapter, we provide detailed methods of use for the recently developed split firefly-based bioluminescence reporter to noninvasively, dynamically, and sensitively
measure ATM kinase activity in live cells and mouse models [7].
2 Materials
1. DNA encoding open reading frame for phospho-proteinbinding domain (e.g., Rad53p FHA2 domain).
2. Full-length coding sequence for the Firefly luciferase (FLuc) or
plasmids coding for the N-terminal luciferase (amino acids
1–416) and C-terminal luciferase (amino acids 398–550)
fragments.
3. Expression vectors with constitutive promoters for expression
in mammalian cells (e.g., pEF vector from Clontech).
4. Optional: Expression vectors and packaging plasmids for
generating lentiviral particles.
5. High fidelity DNA polymerases (e.g., Pfu DNA polymerase),
dNTPs, oligonucleotide primers for cloning and sequencing,
PCR reaction buffers, PCR Thermocycler, restriction endonuclease, DNA ligase, site-directed mutagenesis kits, high efficiency
competent cells, antibiotic, bacterial growth media (LB, SOC),
agar plates, plasmid DNA extraction kits, DNA gel purification,
and sequencing kits.
1. HEK293T cells or other readily transfectable cell lines.
2. Desired cell line(s) for biologic question of interest (i.e.,
D54, U87).
3. Fetal bovine serum (FBS).
4. Complete growth medium with FBS: Growth media with 10%
FBS, and 1% penicillin/streptomycin.
5. Serum-free medium: Growth media without FBS and penicillin/
streptomycin.
6. Trypsinization medium: 0.05% Trypsin-EDTA.
7. 1000× penicillin stock solutions: 10,000 Units/mL penicillin.
8. 1000× streptomycin stock solution: 10 mg/mL streptomycin.
9. 1000× geneticin/G418 stock solution: 50 mg/mL geneticin/
G418.
2.1 Molecular
Biology
2.2 Cell Culture
Shyam Nyati et al.
You, and Hunter [11] described a fluorescence resonance energy
transfer (FRET)-based biosensor for monitoring ATM kinase activity in live cells. Although this reporter provides direct measurement of the ATM kinase activity, it has a limitation of usability in
mouse model due to tissue penetration and autofluorescence in the
CFP-YFP range. In this book chapter, we provide detailed methods of use for the recently developed split firefly-based bioluminescence reporter to noninvasively, dynamically, and sensitively
measure ATM kinase activity in live cells and mouse models [7].
2 Materials
1. DNA encoding open reading frame for phospho-proteinbinding domain (e.g., Rad53p FHA2 domain).
2. Full-length coding sequence for the Firefly luciferase (FLuc) or
plasmids coding for the N-terminal luciferase (amino acids
1–416) and C-terminal luciferase (amino acids 398–550)
fragments.
3. Expression vectors with constitutive promoters for expression
in mammalian cells (e.g., pEF vector from Clontech).
4. Optional: Expression vectors and packaging plasmids for
generating lentiviral particles.
5. High fidelity DNA polymerases (e.g., Pfu DNA polymerase),
dNTPs, oligonucleotide primers for cloning and sequencing,
PCR reaction buffers, PCR Thermocycler, restriction endonuclease, DNA ligase, site-directed mutagenesis kits, high efficiency
competent cells, antibiotic, bacterial growth media (LB, SOC),
agar plates, plasmid DNA extraction kits, DNA gel purification,
and sequencing kits.
1. HEK293T cells or other readily transfectable cell lines.
2. Desired cell line(s) for biologic question of interest (i.e.,
D54, U87).
3. Fetal bovine serum (FBS).
4. Complete growth medium with FBS: Growth media with 10%
FBS, and 1% penicillin/streptomycin.
5. Serum-free medium: Growth media without FBS and penicillin/
streptomycin.
6. Trypsinization medium: 0.05% Trypsin-EDTA.
7. 1000× penicillin stock solutions: 10,000 Units/mL penicillin.
8. 1000× streptomycin stock solution: 10 mg/mL streptomycin.
9. 1000× geneticin/G418 stock solution: 50 mg/mL geneticin/
G418.
2.1 Molecular
Biology
2.2 Cell Culture
Shyam Nyati et al.