A PCR-based high-throughput screen with multiround sample pooling: application to somatic cell gene targeting.
Here, we describe a method of systematic PCR screening with multiround sample pooling for the isolation of rare PCR-positive samples. As an example, we have applied this protocol to the recovery of gene-targeted clones in human somatic cells comprising only 0.02-0.17% of cells transduced with targeting vectors. Initially, cells infected with targeting vectors are seeded and grown in fourteen 96-well tissue culture plates. Samples are then collected from these plates and subjected to two rounds of pooling to yield twelve 'superpools' used for an initial PCR. After identifying PCR-positive samples, de-pooling is carried out with successive rounds of PCR screening, using samples of decreasing complexity. Single-cell cloning is subsequently performed to isolate gene-targeted clones. The entire protocol can be completed in 4-8 weeks depending on the proliferative capacity of the cell line.
Konishi
Hiroyuki
H
Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, 1650 Orleans Street, Baltimore, Maryland 21231, USA. hkonish2@jhmi.edu
Lauring
Josh
J
Garay
Joseph P
JP
Karakas
Bedri
B
Abukhdeir
Abde M
AM
Gustin
John P
JP
Konishi
Yuko
Y
Park
Ben H
BH
D018929:Cell Culture Techniques; D002460:Cell Line; D018390:Gene Targeting; D005822:Genetic Vectors; D006801:Humans; D016296:Mutagenesis; D016133:Polymerase Chain Reaction / Q000379:methods*; D014161:Transduction, Genetic