Letters to the Editor
Am. J. Hum. Genet. 46:635-636, 1990
Lymphoblastoid Cell Lines from Frozen Whole Blood: A Quick and Economical Safeguard for Linkage Analysis To the Editor:
There is considerable debate among human geneticists on the safest and cheapest strategy for obtaining sufficient DNA from an individual for linkage analysis (or indeed for any other project that requires a large but unknown amount of DNA). One approach is to establish a lymphoblastoid cell line (LCL) from isolated white blood cells (Neitzel 1986). While this provides an infinite source of DNA, it is enormously expensive in terms of labor and materials, both at the initial stages of gradient sedimentation of the leukocytes (particularly if many members of a pedigree are bled at once) and during the culture of the lines. Another strategy is to collect sufficient blood (about 50 ml) to have reasonable confidence that by direct isolation it will provide enough DNA for the entire mapping project. However, venipuncture of such volumes can reduce patient cooperation, particularly with small children, and it provides no safeguard should accident befall the DNA extraction or should the sample be used up. We are using a different strategy, based on our method of establishing LCLs from as little as 0.5 ml whole blood. Although we find that the method works with fresh blood, it is particularly convenient that we find that transformation is detected earlier and more easily if frozen blood is used, because this lyses most of the red cells. We therefore make two whole blood freezes i 1990 by The American Society of Human Genetics. All rights reserved. 0002-9297/90/4603-0029$02.00
(0.5 ml each) from each blood sample, and these are kept as a backup in case it is necessary to establish an LCL at a later date. We are then left with enough blood from a 20-ml sample to make approximately 400 ig DNA by using the salting-out method (Miller et al. 1988), which allows us to process 14 samples in 1.5 h (at a cost of less than $1/sample). A similar method, using a more dilute concentration of virus and cells, has been reported elsewhere (Ventura et al. 1988), but was unsuccessful in our hands and has not been commonly accepted, perhaps in part because the value of using frozen cells was not indicated in the report. We have successfully established LCLs from 35/35 whole-blood samples which have been frozen with 10% DMSO for as long as 5 mo. The cells were thawed rapidly and were rinsed in RPMI 1640 before incubation for 2 h at 370C in 0.3 ml filtered Epstein Barr virus-containing supernatant from a B95-8 marmoset culture. Then 0.8 ml RPMI 1640 containing 20% FCS and 2 gli phytohemagglutinin-P/ml was added (Moss et al. 1979). Cells were plated out in a serial dilution in a 96-well titer plate and incubated at 370C with 5% C02 with twice-weekly media changes using RPMI 1640 with 20% serum until transformed colonies were apparent when they were subcultured twice within the 96-well plate, then twice within a 24-well plate before being transferred to culture flasks in which they were maintained on RPMI 1640 with 10%-15% serum. GEORGIA CHENEVIX-TRENCH, BEVERLEY KERR, AND MARILYN WALTERS Queensland Institute of Medical Research
Brisbane 635
636 References Miller SA, Dykes DD, Polesky HF (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16:1215 Moss DJ, Rickinson AB, Pope JH (1979) Long-term T-cellmediated immunity to Epstein-Barr virus in man. III. Activation of cytotoxic T cells in virus-infected leucocyte cultures. Int J Cancer 23:618-625
Letter to the Editor Neitzel H (1986) A routine method for the establishment of permanent growing lymphoblastoid cell lines. Hum Genet 73:320-326 Ventura M, Gibaud A, Le Pendu J, Hillaire D, Gerard G, Vitrac D, Oriol R (1988) Use of a simple method for the EpsteinBarr virus transformation of lymphocytes from members of large families of Reunion Island. Hum Hered 38:36-43
Am. J. Hum. Genet. 46:635-636, 1990
Lymphoblastoid Cell Lines from Frozen Whole Blood: A Quick and Economical Safeguard for Linkage Analysis To the Editor:
There is considerable debate among human geneticists on the safest and cheapest strategy for obtaining sufficient DNA from an individual for linkage analysis (or indeed for any other project that requires a large but unknown amount of DNA). One approach is to establish a lymphoblastoid cell line (LCL) from isolated white blood cells (Neitzel 1986). While this provides an infinite source of DNA, it is enormously expensive in terms of labor and materials, both at the initial stages of gradient sedimentation of the leukocytes (particularly if many members of a pedigree are bled at once) and during the culture of the lines. Another strategy is to collect sufficient blood (about 50 ml) to have reasonable confidence that by direct isolation it will provide enough DNA for the entire mapping project. However, venipuncture of such volumes can reduce patient cooperation, particularly with small children, and it provides no safeguard should accident befall the DNA extraction or should the sample be used up. We are using a different strategy, based on our method of establishing LCLs from as little as 0.5 ml whole blood. Although we find that the method works with fresh blood, it is particularly convenient that we find that transformation is detected earlier and more easily if frozen blood is used, because this lyses most of the red cells. We therefore make two whole blood freezes i 1990 by The American Society of Human Genetics. All rights reserved. 0002-9297/90/4603-0029$02.00
(0.5 ml each) from each blood sample, and these are kept as a backup in case it is necessary to establish an LCL at a later date. We are then left with enough blood from a 20-ml sample to make approximately 400 ig DNA by using the salting-out method (Miller et al. 1988), which allows us to process 14 samples in 1.5 h (at a cost of less than $1/sample). A similar method, using a more dilute concentration of virus and cells, has been reported elsewhere (Ventura et al. 1988), but was unsuccessful in our hands and has not been commonly accepted, perhaps in part because the value of using frozen cells was not indicated in the report. We have successfully established LCLs from 35/35 whole-blood samples which have been frozen with 10% DMSO for as long as 5 mo. The cells were thawed rapidly and were rinsed in RPMI 1640 before incubation for 2 h at 370C in 0.3 ml filtered Epstein Barr virus-containing supernatant from a B95-8 marmoset culture. Then 0.8 ml RPMI 1640 containing 20% FCS and 2 gli phytohemagglutinin-P/ml was added (Moss et al. 1979). Cells were plated out in a serial dilution in a 96-well titer plate and incubated at 370C with 5% C02 with twice-weekly media changes using RPMI 1640 with 20% serum until transformed colonies were apparent when they were subcultured twice within the 96-well plate, then twice within a 24-well plate before being transferred to culture flasks in which they were maintained on RPMI 1640 with 10%-15% serum. GEORGIA CHENEVIX-TRENCH, BEVERLEY KERR, AND MARILYN WALTERS Queensland Institute of Medical Research
Brisbane 635
636 References Miller SA, Dykes DD, Polesky HF (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16:1215 Moss DJ, Rickinson AB, Pope JH (1979) Long-term T-cellmediated immunity to Epstein-Barr virus in man. III. Activation of cytotoxic T cells in virus-infected leucocyte cultures. Int J Cancer 23:618-625
Letter to the Editor Neitzel H (1986) A routine method for the establishment of permanent growing lymphoblastoid cell lines. Hum Genet 73:320-326 Ventura M, Gibaud A, Le Pendu J, Hillaire D, Gerard G, Vitrac D, Oriol R (1988) Use of a simple method for the EpsteinBarr virus transformation of lymphocytes from members of large families of Reunion Island. Hum Hered 38:36-43
