Dear bionetters,
I am doing some experiments to optimise conditions for somatic cell
hybridisation between human myeloid leukemia cells and mouse A9 cells (which
are HPRT -ve fibroblasts and can be killed in HAT medium). My ultimate aim is
to obtain a human x mouse hybrid that is:
a) immortalized
b) contains a derivative chromosome from the human leukemia cells
isolated from its homologous human chromosomes (for gene mapping)
I have been culturing the A9 cell in DMEM with 10% calf serum. In the course
of culturing them, a small percentage of (sometimes multinucleated) giant
cells appear. This occurs even when subcloned, so the giant cells are derived
from the same population as the "normal" A9 cells. Does anyone know the cause
of this? Is this normal? I have traced the history of the A9 line to
carcinogenically transformed mouse fibroblasts (L cells) grown at the NCI in
the 1940's. In an article in JNCI, it merely describes the appearance of the
giant cells without giving any hypothesis regarding their origin. I would be
interested in hearing from anyone with experience in culturing A9's to let me
know of their experiences.
I would also like to hear from anyone with experience in somatic cell fusion
(particularly using A9 or human leukemia or normal white blood cells) using
polyethylene glycol (PEG). The brand I'm using is BDH PEG 1500 at 35% w/v in
serum free RPMI 1640, using the method described in:
"METHODS IN MOLECULAR BIOLOGY VOL.29: Chromosome Analysis Protocols"
Edited: John R Gosden, 1994, Humana Press
Ch 17: 'Immortalized Cell Lines: Their creation and use in gene
mapping' by Veronica van Heyningen.
My specific questions are:
1) Will hybrids between (adherent) A9 and (non-adherent) human hematopoietic
cells be adherent, non-adherent, semi-adherent or combinations of these?
2) How well do hybrids grow compared to the parent cells?
3) When and how quickly should I "wean" the hybrids of the HAT medium?
4) How efficient is the process of fusion when it works well?
5) What determines the rate of chromosome loss / retention for non-selected
chromosomes?
6) How many human vs mouse chromosome does one expect in the hybrid
clones?
Answers to any of these questions (or advice on other related matters) would
be much appreciated.
Thanks
Alberto Catalano e-mail: kanemats at morgan.angis.su.oz.au
Ph: (02) 515-7453
Fax: (02) 515-6255
Kanematsu Laboratories * or * Kanematsu Laboratories
Royal Prince Alfred Hospital Level 3, Blackburn Bldg, D06
Missenden Rd, Camperdown NSW 2050 Sydney University NSW 2006
Australia Australia
