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Human or rat growth hormone (GH) genes have been introduced into all cells of a mouse by microinjection of fertilized eggs but they were not expressed under their own promoters. However, substitution of a mouse metallothionein (MT) promoter allowed expression and regulation comparable to that of the endogenous MT genes. These fusion genes have been used to stimulate the growth of both normal mice and dwarf mice that lack sufficient GH. Substitution of a rat elastase-I promoter directed expression of GH exclusively to the acinar cells of the pancreas. Progress has been made towards developing the hGH gene into a vector that is not expressed in vivo unless an enhancer element is inserted. Recombination between overlapping DNA fragments derived from a MThGH gene, each of which is nonfunctional, has been observed when they are coinjected into mouse eggs. In some cases, functional hGH was produced as evidenced by enhanced growth of the mice.
Growth hormone (GH) is an intermediary in a cascade of hormones that control growth of mammals. It is a single chain polypeptide of 191 amino acids that is synthesized by sommatotroph cells in the pituitary. Hypothalamic hormones, somatostatin, and growth hormone releasing factor, regulate GH synthesis; GH, in turn, regulates the production of insulin-like growth factor-I (IGF-I) by peripheral tissues (Palmiter et al. 1983). GH genes have been isolated from several species (Barta et al. 1981; Seeburg 1982; Gordon et al. 1983). They are composed of five exons and span a total of 2–3 kb; thus, they are of a convenient size for genetic manipulation. They are related to placental lactogen and prolactin genes (Niall et al. 1971).
Several groups have introduced genes into the germline of mice by microinjecting appropriate DNA fragments isolated from plasmids into the pronuclei of fertilized eggs (Gordon et al. 1980; Brinster et al. 1981; Costantini and Lacy 1981; E. Wagner et al. 1981; T. Wagner et al. 1981). With current techniques, about 25% of the mice that develop from this procedure retain the foreign DNA in all of their cells and transmit them to half of their offspring. Here, we summarize the results obtained with GH genes and indicate some of the future directions.
Because of our initial/success in obtaining regulated expression of thymidine kinase gene by fusing the mouse metallothionein-I (MT) promoter to the structural gene isolated from herpes simplex virus (Brinster et al. 1981; Palmiter et al. 1982a), we initiated our experiments with GH in a similar manner. In the first experiments, the MT promoter was fused to the structural gene of rat GH (rGH), and DNA fragments retaining 185 bp of MT promoter and all of the rGH structural gene were microinjected into the pronuclei of fertilized eggs. Of the 21 mice that developed from these eggs, six grew significantly larger than control littermates, and several of these mice had extraordinarily high levels of GH mRNA in the liver and rGH in the serum (Palmiter et al. 1982b). Several lines of mice were started from these transgenic founders. One of these lines, MGH-10, is now in the sixth generation; about 50% of the offspring inherit the chromosome carrying the MTrGH genes and all of these mice grow to about twice the size of normal littermates (Table 1).
These mice grow because the mouse MT promoter causes GH to be synthesized in several organs, notably liver and kidney, instead of in the sommatotroph cells of the pituitary. Although the cellular rate of GH production in these transgenic mice may be lower than in the sommatotroph cells, the enormous size of these organs compared to the pituitary allows serum concentrations to reach levels that are 1000-fold higher than normal (Palmiter et al. 1982b). The production of rGH can be modulated about tenfold by adding zinc, a natural inducer of MT genes, to the diet (Hammer et al. 1984a). However, this extra stimulation of GH synthesis is not required to stimulate growth, presumably because the basal rate of synthesis is sufficient to saturate GH receptors.
One of the original attractions of GH genes for expression in transgenic mice was the existence of several mutant strains of mice with defects in GH production. One dwarf strain, called little, grows to about half normal size when homozygous (lit/lit). Although the primary defect that leads to suboptimal GH production in little mice is unknown, injection of GH will stimulate growth (Beamer and Eicher 1976). Therefore, we reasoned that the phenotypic defects in growth could probably be overcome by introducing MTrGH fusion genes into fertilized (lit/lit) eggs. Table 1 shows that enhanced growth was achieved by using this approach (Hammer et al. 1984b).
Homozygous (lit/lit) males show a high degree of infertility, whereas females eventually reach full fertility. The fertility of male little mice expressing MTrGH genes was also corrected; all transgenic males have sired at least two litters. However, the fertility of females (either lit/lit or wild-type) that express MTrGH genes was impaired (Table 1 and Hammer et al. 1984b).
In humans, there is a cluster of five GH-related genes located within 45 kb of DNA on chromosome 17. These genes have been isolated on two cosmids (Barsh et al. 1983); one of these, cGH4, contains the normal hGH gene, a placental lactogen-like gene (hPLL) and a normal placental lactogen gene (hPLA). None bf sixteen transgenic mice carrying this cosmid showed enhanced growth, serum hPL, or hGH as measured by RIA (Fig. 1). Another 16 transgenic mice with plasmids containing either hGH or rGH genes also failed to express these genes (Hammer et al. 1984b). Thus, it appears that the signals necessary for proper expression of human or rat GH genes are either absent from the DNA molecules tested thus far or they are incapable of responding to mouse regulatory factors when introduced into the germ-line by microinjection.
We have achieved expression of hGHN and hPLA by fusing these structural genes to the mouse MT promoter in a manner similar to that used for rGH (Fig. 1). MThGH fusion genes work as well as MTrGH genes at stimulating the growth of mice (Palmiter et al. 1983). The foreign gene is expressed predominantly in liver, heart, testis, and intestine; but measurable levels of MThGH mRNA are detectable in other tissues as well. The pattern of expression resembles that of the endogenous MT genes (Palmiter et al. 1983).
One line of mice expressing a high level (about 7 µg/ml in the serum) of hPLA has been examined in detail to see if this hormone has any effect upon murine growth or reproductive physiology. The fertility of males and females expressing this gene is normal; fetal and adolescent growth are normal; and maternal behavior and lactation are normal. Thus, we have been unable to help unravel the mystery concerning the physiological role of hPL (Chard 1983).
To help define the DNA sequences involved in cell-specific gene expression, it would be useful to have a gene that is not expressed at all in vivo unless appropriate sequences, so-called enhancer elements, are supplied. If the vector coded for a gene product that had a pronounced physiological effect when expressed in any cell type, then one could test various enhancers in a common vector. Expression could initially be monitored by the physiological effect and then the source of the gene product could be tracked to the cell type of origin. The data presented above suggested that GH genes might be adaptable to this purpose since they were not expressed under their own promoters but produced a readily apparent physiological effect when expressed under control of a heterologous promoter. An advantage of GH over many other secreted hormones is that the only post-translational modification involves removal of the signal pep tide. Thus, it should be possible to produce and secrete functional GH in any cell type with signal peptidase.
Our approach was to introduce DNA fragments containing enhancer function upstream of the hGH promoter. We chose the mouse MT-I gene region located between −46 and −185, which includes the entire cluster of metal regulatory elements (MREs), as a test enhancer (Stuart et al. 1984). The various constructs were tested in tissue culture cells by standard transfection and transient assay or by microinjecting them into fertilized eggs and testing for expression in the liver of fetuses or adults. Figure 2 shows that the tissue culture assay was somewhat permissive and allowed a low level of expression (measured as hGH mRNA) whereas the in vivo test system was more stringent and no expression could be detected in the absence of the test enhancer. Insertion of the MRE enhancer region (Fig. 2, solid box) about 300 bp upstream of the hGH cap site had no effect. Likewise, several other enhancers, including the SV40 72-bp repeats, had no effect in vivo when inserted at −300 (data not shown). However, when the MRE region was moved to within 90 bp of the hGH cap site, then expression in tissue culture cells could be detected and hGH mRNA was inducible by metals. However, this construct was still ineffective in vivo unless the vector sequences were removed. The control (hGH−90) without vector sequences is inactive in vivo. We are in the process of testing other enhancers to see if this construct has general utility as an enhancer vector. It appears that DNA sequences lying between −90 and −300 of the hGH gene prevent MRE enhancer activation of hGH transcription and that this gene is very sensitive to vector (pUC) sequences in vivo.
The rat elastase-I gene is expressed in the acinar cells of the pancreas. When this gene was introduced into the germline of mice, rat elastase was still expressed almost exclusively in the pancreas (Swift et al. 1984). Deletion of all but 205 bp of 5′ flanking sequences still allowed pancreas-specific expression but the absolute level of expression was somewhat lower (Fig. 3). To determine whether 5′ rat elastase sequences were sufficient to direct expression to the pancreas, we inserted a convenient linker at +8 of rat elastase gene and fused the 5′ flanking sequences to the +2 position of hGH. This fusion gene was also expressed in a pancreas-specific manner when either 4500 bp or 205 bp of rat elastase sequences were present (Fig. 3). The level of expression of either rat elastase or hGH was at least three orders of magnitude higher in pancreas than in any other tissue tested and in many cases it was five orders of magnitude higher (Swift et al. 1984; Ornitz et al. 1985). Furthermore, the level of foreign mRNA produced frequently exceeded 104 molecules/cell. We do not yet know whether the enhancer and promoter functions are separable, but if so then it appears that the elastase promoter is very tightly controlled by its associated enhancer.
It is interesting to note that the transgenic mice expressing the elastase-hGH fusion genes did not show any signs of enhanced growth despite the high level of expression in the pancreas. Immunofluorescent analysis of hGH in sections of the pancreas from these animals revealed intense fluorescence over the acinar cell’s and in the collecting ducts; but islets, lymph nodes, and capillaries were negative (Ornitz et al. 1985). Thus, we suspect that hGH was secreted along with the digestive enzymes into the gut and none was resorbed intact or secreted into the circulation. In fact, the absence of a growth effect argues strongly that these mice were not synthesizing hGH in any tissue that secretes into the bloodstream. This is one limitation of hGH as the ideal enhancer vector as discussed in the previous section.
One of our goals is to obtain some control over the integration of foreign DNA. At present we have little control over the number of copies that integrate or the site at which they integrate. The integration frequency is improved about fivefold by injecting linear molecules compared to circular forms (Brinster et al. 1985). There is usually a single integration site and if there is more than one copy of the foreign DNA integrated they are usually in a tandem head-to-tail array. The tandem arrays could result from homologous recombination between the injected molecules either before or during the integration process (Brinster et al. 1981). However, we have not observed homologous recombination between the injected DNA and homologous endogenous genes (R. Palmiter et al., unpubl.).
We constructed the MThGH vectors shown in Figure 4 as a means of studying recombination. First, we inserted a FLP sequence from the 2-micron circle of yeast into the third intron of MThGH (Fig. 4, solid circle) because an enzyme (FLPase) that will promote site-specific recombination at this sequence can be isolated (Cox 1983; Meyer-Leon et al. 1984). This enzyme may ultimately be useful for targeting foreign DNA to specific sites. Insertion of the FLP sequence had no effect on MThGH expression (Fig. 4). Then we deleted several hundred base pairs between NarI and SmaI (plasmid #165), which should result in a truncated hGH protein of 104 amino acids. We have not yet looked for this protein, but we know that mRNA levels are high and that the mice do not grow larger than normal; we suspect that this protein is made but is biologically inactive. The ultimate experiment will be to supply the correct information to replace the deleted nucleotides by introducing DNA fragment #193 along with FLPase into eggs from mice carrying resident copies of fragment #165. As a control for this experiment, we coinjected fragments #165 and #193. Ten mice resulting from this experiment retained these plasmid sequences. Southern blots of their DNA revealed the presence of a 1.2 kb PvuII fragment that is indicative of recombination between the two DNA molecules. Five of these mice had mRNA. sequences corresponding to the region deleted from fragment #165, and two of them grew significantly larger than their control littermates. Recombination between DNA molecules that are introduced into cells simultaneously has been reported (Folger et al. 1982; DeSaint Vincent and Wahl 1983; Shapira et al. 1983; Small and Scangos 1983; Subramani and Berg 1983), but in most of those experiments there was selection for the recombination event. In this experiment there was no selection, yet the frequency of recombination was about 70%. Furthermore, in two of these cases the recombination occurred in a manner that allowed production of functional hGH. In the other three mice that showed evidence of gene expression but failed to grow larger than normal, the recombination events may have been imprecise and thereby resulted in an aberrant hGH protein.