Cell, Vol 35, 647-655, December 1963 (Part 2), Copyright 0 1963 by MIT 0092.8674/83/130647-09 $02.00/O Prenatal Lethalities in Mice Homozygous for Human Growth Hormone Integrated in the Germ Line Gene Sequences Erwin F. Wagner,* Luis Covarrubias, Timothy A. Stewart,+ and Beatrice Mintz Institute for Cancer Research Fox Chase Cancer Center Philadelphia, Pennsylvania 19111 Summary The mutagenic potential of recombinant DNA in the germ line was investigated in the descendants of mice obtained from eggs injected with the human growth-hormone gene in a pBR322 vector. Six posi- tive animals (including one mosaic) were produced and had l-20 copies of the donor sequences in unique and often complex integration patterns indic- ative of a single or an interrupted insertion. All were heterozygotes and transmitted the foreign insert to their progeny, forming six new HUGH strains. Mat- ings between heterozygotes yielded viable healthy homozygotes in four of the strains. However, in the HUGH/3 and HUGH/4 strains, no postnatal homozy- gotes were found and litter sizes at birth were small. These two independent cases of mutation, both homozygous recessive prenatal lethals, are attrib- utable to disruption of native sequences by alien ones. They constitute the first instances of inser- tional mutagenesis due to integration of recombinant DNA in the germ line of the mouse. The mutants provide new possibilities for molecular identification of gene functions necessary for normal develop- ment. Introduction The introduction of recombinant DNA into mice by microin- jection into the fertilized egg allows a defined gene to be integrated into the genome at the outset of development and often to be included in the germ line (Gordon et al., 1980; Gordon and Ruddle, 1981; Costantini and Lacy, 1981; E. Wagner et al., 1981; Stewart et al., 1982; Brinster et al., 1981; Palmiter et al., 1982a, 1982b). A recombinant germ line gene may be used in two different experimental strategies to examine the same problem-the molecular basis for developmentally regulated gene expression. The direct strategy is to achieve developmentally appropriate expression of the alien sequences and to relate specific modifications in gene design to corresponding modifica- tions in expression; this objective has yet to be realized. The indirect strategy, which is the chief subject of the present report, is to use the vector as a virtually nonspecific intruder capable of disrupting the regulated expression of a native gene by insertion into or near the native se- * Present address: European Molecular Biology Laboratory, 6900 Heidel- berg, Germany. ‘Present address: Department of Human Genetics, Harvard MedIcal School, Boston, Massachusetts 02115. quences. With the inserted sequences as a probe, it should be possible to recover and characterize the endogenous mutant gene, which in turn becomes a probe for isolation of the normal allele, and to identify the molecular prereq- uisites for normal function. Insertional mutgenesis by mobile genetic elements has long been recognized as a spontaneous phenomenon in maize (McClintock, 1956) and has recently been described in some other eucaryotes, including yeast (Roeder and Fink, 1980) and Drosophila (Kidwell et al., 1977). The cloned transposable elements in Drosophila now serve as vectors in successful gene transfer experiments (Spradling and Rubin, 1982; Rubin and Spradling, 1982). DNA trans- formation has also been shown to be capable of disrupting a gene in yeast (Shortle et al., 1982). In mice, the only known instances of insertional mutagenesis in the germ line have been due to murine leukemia retroviruses. Mu- tation in one case resulted from spontaneous insertion of proviral DNA sequences in the dilute coat color locus (Jenkins et al., 1981) and in the other there was an ex- perimental insertion in the al(l) collagen gene after injec- tion of virus into a postimplantation embryo (Jaenisch, 1980; Schnieke et al., 1983). We describe here the first instances of insertional mu- tagenesis due to integration of recombinant DNA in the germ line of the mouse. The foreign sequences consisted of the human growth-hormone gene in a pBR322 plasmid. Two independent mutations were found-a high incidence among six cases with unique integration patterns of the inserted DNA. Both mutations are recessive prenatal le- thals. These mutants may provide new opportunities for molecular dissection of gene functions required for devel- opment. Results Production of Mice with the Human Growth-Hormone Gene Approximately 6,000 copies of the phGH plasmid (Figure 1) were injected into a pronucleus of (C3H x C57BL/6)F1 fertilized eggs. Of 229 injected eggs, 143 (62%) survived injection and were surgically transferred (along with un- manipulated Icr eggs) into ten pseudopregnant Icr females. Twenty animals were born from the experimental eggs. At approximately 6 weeks of age, partial splenectomy was performed and the DNA was isolated for Southern blot analysis. After digestion with Eco RI and hybridization to DNA of the entire plasmid, six mice (30% of those born from experimental eggs), including five males and one female, proved to be positive for the vector sequences (Figure 2). Each carried at least one copy of the 2.6 kb genomic human growth-hormone gene. The six positive animals became the founders of six new strains designated HUGH/l through HUGH/G. In this report, the genotypes of individuals containing sequences from the plasmid are given as HGH/+ for heterozygotes and HGH/HGH for homozygotes, irrespective of their