Somatic expression of herpes thymidine kinase in mice following injection of a fusion gene into eggs

Abstract

A plasmid, designated pMK, containing the structural gene for thymidine kinase from herpes simplex virus (HSV) fused to the promoter/regulatory region of the mouse metallothionein-I gene, was injected into the pronucleus of fertilized one-cell mouse eggs; the eggs were subsequently reimplanted into the oviducts of pseudopregnant mice. The first experiment produced 19 offspring, one of which expressed high levels of HSV thymidine kinase activity in the liver and kidney. pMK DNA sequences were detected in equal amounts in several tissues of the expressing mouse as well as in three mice that did not express HSV thymidine kinase activity. In all cases, several copies of the pMK plasmid were tandemly duplicated and integrated into mouse DNA. It appears as though multiple copies of the intact plasmid were fused by homologous recombination either before or after integration at a single site in the mouse genome. The overall efficiency of obtaining somatic expression of thymidine kinase in experiments performed to date is about 10% (4/41), and twice this number have integrated pMK DNA. This procedure not only provides a means of introducing new genes into mice, but it will also be a valuable system for studying tissue-specific regulation of gene expression.

Figure 1. Structure of the Plasmids and DNA Fragments Used in this Study

(A) Plasmid pMT-TK was constructed from plasmid m₁pEE_3.8 (Durnam et al.. 1980), which contains a 3.8 kb genomic Eco RI fragment that includes the MT-I gene inserted into the Eco RI site of pBR322, by insertion of the 3.5 kb Bam HI fragment of Herpes Simplex Virus Type I containing the thymidine kinase gene (McKnight, 1980) into the Bam HI site. The two genes are present in the same transcriptional orientation, as shown by the arrows. (B) The fusion plasmid, pMK, was created by digestion of pMT-TK with Bgl II restriction endonuclease followed by ligation with T4 DNA ligase to directly join the 5′ region of the MT-I gene to the TK structural gene. pBR322 sequences are shown by a single line, TK gene sequences by a narrow box and MT-I gene sequences by a wide box. mRNA coding regions are represented by closed boxes; nontranscribed and intron regions are shown by open boxes. Hatched boxes inside the circles represent regions of these genes that ware used as hybridization probes; the MT-I specific probe, MT-XH, extends from Xba I to Hind III and TK-specific probe, TK-BP, extends from Bgl II to Pst I. The fusion plasmid probe, pMK(-EK), includes the entire plasmid except the Eco RI to Kpn I region because Southern blots revealed that this is a sequence present many times in the mouse genome. Restriction sites relevant to the construction of the plasmids and the gene-specific probes are shown in panel A. All restriction sites used in mapping integrated copies of pMK are shown in panel B. pMK is not cut by Hind III, Xba I or Xho I. Also shown are the locations of the TATAA promoter sequences and ATG translation start codons for the two genes.

Figure 2. pMK Sequences Are Present in Four Mice

DNA (6.5 µg) from the kidney (K) of each of the twelve mice, as well as DNA (6.5 µg) from liver (L) and brain (B) of mice 23-1 and 23-2, was digested with restriction endonuclease Bst EII (see Figure 1B), subjected to electrophoresis on a 1.2% agarose gel, transferred to nitrocellulose, and hybridized to a mixture of MT-XH and TK-BP probes (see Figure 1A). The 6 kb band present in all samples represents the endogenous MT-I gene; the 2.3 kb band comigrates with the band generated by Bst EII digestion of pMK. We have noted a decreased intensity of the MT-I band in liver and kidney of mouse 23-2 in several experiments with these DNA preparations, but we cannot provide a satisfying explanation for this result.

Figure 3. Junction Fragments and Tandem Repeats of pMK

(A) Liver DNA (12 µg) from mouse 23-2 was digested with restriction enzymes that cut pMK twice (Bst EII, Eco RI, Pvu II), once (Bam HI, Bgl II, Kpn I), or not at all (Hind III, Xba I), subjected to electrophoresis on a 0.8% agarose gel, transferred to nitrocellulose, and hybridized with a pMK(-EK) nick-translated probe (see Figure 1B). The inset at the bottom of A reveals the major bands more clearly; it was obtained after a 14 hr exposure compared to 60 hr for the main figure. Restriction fragments predicted from the endogenous MT-I gene (⊳) and from cleavage within the repeats of pMK (►) are indicated; the other bands are presumably junction fragments containing both pMK and mouse DNA. (B) Comparison of Pvu II digests of liver, testis and muscle DNA (12 µg each) from mouse 23-2. Markers are a Hind III digest of phage λ.

Figure 4. Models for Integration of pMK DNA into Mouse DNA

The plasmid is illustrated as a circular DNA molecule with two unique restriction sites shown symbolically (). (A) A single plasmid DNA molecule recombines (either by homologous or nonhomologous recombination) with mouse DNA to give a single integrated plasmid. The junction between mouse and plasmid sequences might occur at any site in both plasmid and mouse DNAs. Digestion with either of the two enzymes whose sites are shown would be expected to generate two products, neither of which would be likely to be the same size as linear pMK DNA. (B) After the initial integration event, a number of subsequent events could occur involving homologous recombination of additional copies of pMK into those already integrated, giving rise to a tandem repetition of the integrated pMK sequences. Digestion with either enzyme would generate several copies (three as drawn) of full-length linear pMK molecules, plus single copies of two new junction fragments. (C) Several copies of the plasmid could homologously recombine with one another to generate a tandemly repetitive plasmid, which would then recombine with mouse DNA, again generating a tandemly repeated integrated plasmid. Restriction enzyme analysis would generate the same products as model B.

Figure 5. Junction Fragments and Tandem Repeats of pMK Are Present in All MK-Positive Mice

Liver DNA (10 µg) of all four mice that contain pMK sequences (19-2, 21-3, 23-1, 23-2) and from a control mouse (19-l) was digested with Bam HI, Bgl II and Kpn I (each of these enzymes cuts pMK at a single site: see Figure 1B), subjected to electrophoresis on a 0.8% agarose gel, transferred to nitrocellulose and hybridized to pMK(-EK). Bands common to the 19-1 control and to the other mice come from the endogenous MT-I gene. The major band comigrates with linearized pMK. Additional bands that are presumed to be junction fragments are also observed.

Figure 6. pMK Sequences Are Integrated into Cellular DNA

DNA (16 µg) from the livers of mice 19-1 (control), 23-2, and 19-2 were subjected to electrophoresis on a 0.6% agarose gel without restriction nuclease digestion, after digestion with a mixture of Hind III, Xba I and Xho I (none of these enzymes cuts within pMK), or after digestion with the above enzymes plus Bam HI (which cuts pMK at a single site). (A) shows the ethidium bromide stained gel; (B) shows an autoradiogram after transfer to nitrocellulose and hybridization to a pMK(-EK) probe. The size markers are intact λ DNA and λ DNA digested with Hind III. The strong band seen after Bam HI digestion comigrates with pMK that has been similarly digested.