Transfer of the adenosine deaminase (ADA) gene of a B-lymphoblastoid cell line (LCL) to an ADA-deficient LCL by a microcell-mediated chromosome transfer technique.
As a model of somatic cell gene therapy, a normal adenosine deaminase (ADA) gene was introduced into a B-lymphoblastoid cell line (LCL) established from a patient with ADA deficiency by microcell-mediated chromosome transfer (MMCT). A LCL derived from his mother was used as a gene donor. Seven fusion experiments were performed and hybrid cells were pipetted into 123 wells. After selection in the presence of deoxyadenosine, cells grew in 12 wells at Week 9 after fusion. Among these wells, ADA activity of hybrids was low in 4 wells, 130-280% of that of the donor LCL in 7 wells and very high in one well. Hybrid cells in 4 wells with ADA-positive cells were investigated for the time-course of expression of ADA activity. In one well, ADA activity was expressed until Week 36, while, in 3 wells, ADA activity decreased or was lost between 21-36 weeks after fusion. These findings indicate the transfer of chromosome 20 containing a normal ADA gene into recipient cells and the deletion of this chromosome from some part of the hybrid cells. Karyotyping at Week 35 or 37 revealed 47 chromosomes in about 30% of the cells in 2 wells, which suggests that these hybrids were relatively stable in culture.
Sawami
H
H
Ito
K
K
Norioka
M
M
Monden
S
S
Fujita
M
M
Uchino
H
H
D000243:Adenosine Deaminase / Q000172:deficiency / Q000235:genetics*; D001402:B-Lymphocytes; D002478:Cells, Cultured; D002890:Chromosomes, Human, Pair 20; D002896:Chromosomes, Human, Pair 6; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D009700:Nucleoside Deaminases / Q000235:genetics*; D014162:Transfection / Q000235:genetics*
D009700:Nucleoside Deaminases; D000243:Adenosine Deaminase