TIG - - May 1987, Vol. 3, no. 5

The real reverse genetics: targeted mutagenesis in the mouse lan J. Jackson MRC Clinical and P@ulation Cytogenetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK.

The mouse has long been the mammalian geneticist's best friend. Decades of study and countless millions of animals have provided us with an excellent genetic map, until recently undoubtedly the best for any manunal. In the last few years, however, the huge effort in human genetics has resulted in so many genes and cloned DNA fragments being placed on the human map that, in some aspects at least, it now rivals the mouse map.

The mouse remains the choice for experimental developmental stud- ies, but what role is there for the mouse in medical genetic research? There is a considerable one if the outcome of identifying and cloning human genetic disease genes is to lead not only to prenatal diagnosis and termination but also to a better understanding and treaUnent of the disease itself. The normal expres- sion of particular genes can be studied more easily during mouse embryogenesis than during human development. More important, however, are mouse models for genetic disease, in which the conse- quences of abnormal expression can be investigated, and prospective treatments tested.

Until now the identification of these mouse parallels to human disorders has been a matter of looking at mouse mutants and seeing what was there. Two very significant papers published recently in Nature 1"~ promise to change that approach. The work, by Michael Kuehn and co-workers in Cam- bridge, and independently by Martin Hooper of Edinburgh University and collaborators, reports the directed production of mouse models for human Lesch-Nyhan Syndrome. Lesch-Nyhan is a fortunately rare but very serious disorder of man, caused by a deficiency of the X-linked enzyme hypoxanthine-guanine phos- phoribosyltransferase (HPRT). The disease, affecting almost exclusively

males, is characterized by motor defects, impulses to self-mutilation and other neurological disorders. It is not known how the defect in purine metabolism causes the symptoms.

No naturally occurring nnimal models for the disease have so far been identified. The Edinburgh and Cambridge groups now report the use of embryonal stem cells (ES or EK cells) to produce HPRT-deficient mice. ES cells are derived from early mouse embryos and were first described by Evans and Kaufman in Cambridge s. They can be grown in culture for considerable periods, and subsequently reintroduced into mouse blastocysts, where they con- tribute to the developing embryo to

Embryo

Pluripotent ES cells

HPRT - deficient ES cells

ch|maera

monito produce a chimaeric animal. These mice are made up of two cell populations, the host embryo and the donor ES cells, which are usually distinguished from each other by a difference in pigmentation pheno- type. ES-derived ('.ells are found in all tissues of the body, including the gonads from where they are passed on through the germ line. (A review by E. Robertson ,of the Cambridge group has appeared in TIG4.) In essence, to produce HPRT-deficient mice both groups selected in culture for HPRT-deficient ES cells, intro- duced them into host embryos, and bred from the resultant chimaeras (see Fig. 1). It is a simple concept, and indeed it is not new. Beatrice Mintz and colleagues attempted the same thing over t-0 years ago s, but were not successful, mainly because they were using embryonal carci- noma (EC) cells which rarely, ffever, colonize the germ line. The succes- ses now reported are indicative of the foresight and persistence of the Edinburgh and Cambridge groups in developing the ES-celllines and their considerable technical skills in exploiting them.

~) 1987, I ~ P u b l i c , Celllhfidge 0168 - 9 5 2 5 / ~ . 0 0

Mutagenesis m culture I

I Selection lor HPRT deficiency I

I Introduclion of ES cells I into a host embryo I

Germ line ] formation

Analysis of " - 'transgenlc' f, I

Fig. 1. Producing HPRT-defici~,nt mice fTom cultured embryonal stern (E$) cells.

n i t o r Both groups began with male ES

cells, which carry only one Hprt gene on their single X chromosome. The Cambridge group had shown previously s that mass, multiple in- fecfion of the cells with a retroviral vector results in up to 20 viral DNA insertions into each cell's genome; they have furthermore used these cells to generate a number of mice transmitting many of these novel integrants through their germ line. These should incidentally prove a valuable source of insertional muta- tions, each one tagged by the retrovirus. From such a mutagenized population Kuehn et al. identified HPRT-deficient cells by selection on 6-thioguanine (6-TG). Southern blot- ting revealed that in the two cases examined the mutation was caused by a retroviral insert into introns of the Hprt gene. Hooper et al. used 6- TG selection of spontaneous muta- tions to obtain their deficient cells. The cells were then introduced into blastocyst embryos.

The ES cells are male, but are introduced into host embryos of either sex. [f they are introduced into male embryos, both host and donor cells may contribute to the sperm. However, if they are in a female host, providing that the XY donor contribu- tion is sufficiently high, the XX embryo is converted to a phenotypic male. As XX germ cells will not form sperm, all sperm from these animals will be of the donor type, and all X- bearing sperm will transmit the mutant Hp~ gene. This is exactly what both sets of workers report. In addition, Kuehn et al. also find that one of their ES lines has lost its Y chromosome, making it XO. Such chromosome loss may be quite common after clonal selection of ES cells. They see no conversion of XX embryos to males with this fine, but the X0 cells contributed to the germ line of a female chimaera.

The chimaeric males transmit the mutant Hprt gene to their female offspring, which are carriers of the mutation. They will all be mosaic with respect to HPRT activity due to random inactivation of the normal or the mutant X chromosome. Hooper etal. examined these carrier females by measuring the HPRT/APRT enzyme activity ratio in single hair follicles, and showed them to be lower than the normal control range. (This test is used on humans to

1 determine Lesch-Nyhan carrier sta-

t~ls.) They also looked at the ratio in single intestinal crypts, which are clonal in origin, and as expected, they fall into two classes: those with normal levels of HPRT and those with very low or undetectable levels. Kuehn et al. used a very sensitive radioactive assay to show that pri- mary cell cultures derived from putative carrier females contained two classes of cells, with normal HPRT activity or with none.

Carrier females were mated, and transmitted the mutation to their offspring. In addition, Kuelm et al. were able to breed from the X0- containing female chimaera, and in this way directly obtained male mice hemizygous for the mutation. All three mutant alleles are established as breeding lines. Enzyme-deficient male progeny of these lines are the mouse equivalent of Lesch-Nyhan human males. The mutation is not lethal, even though the sensitive autoradiographic assay detects no HPRT activity in these animals. It remains to be established whether the mice display some of the symp- toms of Lesch-Nyhan syndrome. Human Lesch-Nyhan patients do not display symptoms at birth, are de- velopmentally retarded at a few months but often do not show the serious neurological abnormalities until two years of age or later. It is reported that so far, up to 12 weeks, none of the HPRT-deficient mice have any obvious gross abnormality, neurological or otherwise.

It must be said that mice and men are clearly different, and there may well be differences in the manifesta- tions of equivalent genetic mutations (perhaps in particular in neurological disorders where human develop- ment is presumably more complex, and our ability to discern abnormali- ties is sharper). Nevertheless, these studies represent the first instance of directed mutation in the mouse.

TIG - - M a y 1987, Vol. 3, ~ . 5

What everyone hopes now is that the next step will work; that is, pro- ducing mutations in genes for which there is no direct selection. The best bet for this is to reproduce in ES cells the homologous recombination that the groups of Smithies and Capecchi 7's have demonstrated in other cultured cells. DNA introduced into the stem cells might be made to recombine homologously with one of its endogenous complements, and in doing so eliminate its function. Probes from putative human disease loci might be used to produce mutations in the mouse and allow the identity of these clones to be proved. It should be possible to produce mouse models for, say, muscular dystrophy or cystic fibrosis. More exciting still, it will be possible to produce completely new mutations by disrupting genes whose function is unknown, but which are expected to play a role in development; the homeobox genes, for example. Some of us will not be content simply to await results.

A c k n o w l e d g e m e n t s I thank the Lister Institute of

Preventive Medicine for financial support.

References I Kuehn, M. R., Bradley, A., Robertson,

E. J. and Evans, M. J. (1987) Nature 326, 295-298

2 Hooper, M., Hardy, K., Handyside, A., Hunter, S. and Monk, M. (1987) Nature 326, 292-295

3 Evans, M. J. and Kaufinan, M. H. (1981) Nature 292, 154--156

4 Robertson, E. J. 0986) Trends Gemet. 2, 9-13

5 Dewey, M. J., Martin, D.W., Martin, G. R. and Mintz, B. (1977)Proc. Natl Acad. Sci. USA 74, 5564-5568

6 Robertson, E. J., Bradley, A., Kuehn, M. and Evans, M. (1986) Nature 323, 445-448

7 Srmthies, O. etal. (1985) Nature 317, 230- 234

8 Thomas, K. R., Folger, K. R. and Capecctd, M. R. (1986) Cell 44, 419-428

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