.=/ 1992 Oxford University Press

Nucleic Acids Research, Vol. 20, No. 14 3795

A new mouse embryonic stem cell line with good germ line contribution and gene targeting frequency Thomas M.Magin, Jim McWhir and David W.Melton Institute of Cell and Molecular Biology, University of Edinburgh, Darwin Building, King's Buildings, Mayfield Road, Edinburgh EH9 3JR, UK Submitted May 5, 1992 The use of gene targeting in embryonic stem (ES) cells provides an attractive method for the introduction of genetic alterations into the mammalian germ line (for review see 1). For this ES cell system to operate efficiently the cell lines used should ideally show a high frequency of gene targeting and should contribute readily to the germ line of chimaeric animals. Our early gene targeting experiments were carried out in the hypoxanthine phosphoribosyltransferase (HPRT) deficient ES cell line, El4TG2a (2). This line was isolated as a spontaneous HPRTdeficient derivative of the wild-type E14 line, which was obtained from strain 129 blastocysts. The HPRT deficiency in El4TG2a resulted from a deletion which removed the 5' end of the gene and upstream sequences (3). We used a gene targeting vector to correct the deficiency and obtained a low frequency of transmission of the corrected allele in chimaeric mice produced by injecting HPRT-corrected ES cells into host blastocysts (3). Because of the origin of the El4TG2a line from wild-type ES cells and the culturing required to generate and identify corrected cells, our blastocyst injections had been inevitably carried out with late passage cells (pass. 36). It has been argued that prolonged in vitro culture is undesirable because it increases the probability that genetic and epigenetic alterations may accumulate in the ES cell population and reduce the capacity to populate the germ line (4). For alternative gene targeting strategies and improved germ line transmission frequencies, we isolated a new ES cell line, HM-1 (5), directly from HPRT-deficient strain 129 mice that had been produced by blastocyst injection of E14TG2a cells (2).

The germ line contribution data for the two HPRT-deficient lines are summarised in Table 1. 60% of all the male chimaeras produced with HM-1 cells transmitted the ES cell-derived coat colour markers and 90% of the fertile chimaeric males showed germ line transmission. This confirmed the hypothesis about the capacity to populate the germ line since, at the same time less than 5% of the E14TG2a chimaeras transmitted the ES cell genome. The gene targeting frequencies in E14TG2a (passage 33) and HM-1 (passage 9) cells were also compared. Table 1 shows that the standard plating efficiencies, the transfection frequencies (as determined by uptake and expression of an HPRT minigene) and the gene targeting frequencies (as determined using an HPRT correcting vector) were essentially identical. Thus, we see no evidence that prolonged in vitro culture produces ES cell populations with an altered capacity for homologous recombination. It should be emphasised that all ES culture was carried out in the absence of feeder cells on gelatin-coated dishes and in medium supplemented with recombinant LIF (6). The new HM-1 line combines a very high degree of germ line contribution, characteristic of early passage ES cells, with the gene targeting frequency characteristic of a more established line which is losing its ability for germ line contribution. In addition to conventional marker genes used in established ES cell lines, the new line permits the use of HPRT as another selectable gene. More importantly, as HPRT can be selected for and against, two-step gene targeting procedures designed to introduce subtle gene alterations (7) can be carried out in HM-1 cells using HPRT minigenes as the sole selectable marker.

Table 1. Comparison of gene targeting and germ line contribution frequencies for two HPRT-deficient ES cell lines Line

El4TG2a HM-1

Germ line contribution1

number2

Plating efficiency (%)3

frequency4

frequency'

2/45 9/15

33 9

18 14

114 117

5.8 5.9

Passage

Transfection

Gene targeting

'Proportion of male chimaeras showing transmission of ES cell-derived gametes, as determined by coat colour markers in test crosses. E14TG2a chimaeras were generated with cells ranging from passage 31 -46. HM-1 chimaeras were generated with cells ranging from passage 5-21. There was no correlation between passage number and degree of germ line contribution for HM-1 cells. 2Cumulative passage number (since the isolation of ES cell lines from blastocysts) at which the plating, transfection and gene targeting experiments were carried out. 3250 cells were plated in 25 cm2 flasks and the number of colonies formed was scored 7 days later. The plating efficiencies (%) shown are the average from two separate determinations. 45 x 106 cells were electroporated with 50 Ag DNA from the HPRT minigene PGK/pDWM1 (5) and plated into a 90 mm dish. HAT selection for HPRT expression was imposed 24 hours later and the surviving colonies were scored after 10 days. The transfection frequency is given as the average number (from two separate determinations) of surviving colonies/dish. 55 x 106 cells were electroporated with 20 yg DNA from the HPRT correcting vector pDWM 102 (3) and plated into a 90 mm dish. Colonies surviving HAT selection were scored after 10 days. The gene targeting frequency is expressed as the average number (from 10 separate determinations) of surviving colonies/dish.

3796 Nucleic Acids Research, Vol. 20, No. 14

ACKNOWLEDGEMENTS T.M.M. is supported by a research fellowship from the D.F.G. J.McW. is a C.R.C. postdoctoral research fellow. This work was supported by the Cancer Research Campaign.

REFERENCES 1. Magin,T.M. and Melton,D.W. (1992) In Rosenberg et al. (eds), 7he Molecular and Genetic Basis of Neurological Disease. ButterworthHeinemann, London, in press. 2. Hooper,M., Hardy,K., Handyside,A., Hunter,S. and Monk,M. (1987) Nature

326, 292-295. 3. Thompson,S., Clarke,A.R., Pow,A.M., Hooper,M.L. and Melton,D.W. (1989) Cell 56, 313-321. 4. Yagi,T., Ikawa,Y, Yoshida,K., Shigetani,Y., Takeda,N., Mabuchi,I., Yamamoto,T. and Aizawa,S. (1990) Proc. Natl. Acad. Sci. USA 87, 9918-9922. 5. Selfridge,J., Pow,A.M., McWhir,J., Magin,T.M. and Melton,D.W. (1992) Somatic Cell and Mol. Genet. in press. 6. Smith,A.G. (1991) J. Tiss. Cult. Meth. 3, 89-94. 7. Hasty,P., Ramirez-Solis,R., Kninlauf,R. and Bradley,A. (1991) Nature 350, 243-246.

A new mouse embryonic stem cell line with good germ line contribution and gene targeting frequency.

=/ 1992 Oxford University Press Nucleic Acids Research, Vol. 20, No. 14 3795 A new mouse embryonic stem cell line with good germ line contribution...
237KB Sizes 0 Downloads 0 Views