A trial of somatic gene targeting in vivo with an adenovirus vector

Abstract

Background: Gene targeting in vivo provides a potentially powerful method for gene analysis and gene therapy. In order to sensitively detect and accurately measure designed sequence changes, we have used a transgenic mouse system, MutaMouse, which has been developed for detection of mutation in vivo. It carries bacteriophage lambda genome with lacZ+ gene, whose change to lacZ-negative allele is detected after in vitro packaging into bacteriophage particles. We have also demonstrated that gene transfer with a replication-defective adenovirus vector can achieve efficient and accurate gene targeting in vitro.

Methods: An 8 kb long DNA corresponding to the bacteriophage lambda transgene with one of two lacZ-negative single-base-pair-substitution mutant allele was inserted into a replication-defective adenovirus vector. This recombinant adenovirus was injected to the transgenic mice via tail-vein. Twenty-four hours later, genomic DNA was extracted from the liver tissue and the lambda::lacZ were recovered by in vitro packaging. The lacZ-negative phage was detected as a plaque former on agar with phenyl-beta-D-galactoside.

Results: The mutant frequency of the lacZ-negative recombinant adenovirus injected mice was at the same level with the control mouse (approximately 1/10000). Our further restriction analysis did not detect any designed recombinant.

Conclusion: The frequency of gene targeting in the mouse liver by these recombinant adenoviruses was shown to be less than 1/20000 in our assay. However, these results will aid the development of a sensitive, reliable and PCR-independent assay for gene targeting in vivo mediated by virus vectors and other means.

Figure 1

Experimental steps to detect gene targeting in vivo. Gene targeting in vivo in liver cells was attempted after the delivery of donor DNA with an adenovirus vector. The gene with the required sequence change (lacZ^-) on the lambda transgene in the mouse will be detected after its recovery in bacteriophage particles. Only lacZ-negative mutants can form plaques under the selective conditions.

Figure 2

Construction of the recombinant adenovirus AdNY58. The bacteriophage lambda LIA7 was recovered from the MutaMouse by in vitro packaging. An SmaI-SacI fragment of LIA7 within its lacZ gene was inserted into pIK153. The Tyr105Stop mutation (Figure 3) was introduced into the resulting plasmid (pIK153LZS.6) using site-directed mutagenesis by PCR as follows. The PCR products generated with the primer pair LZT-U (5'-CGAAGAGGCCCGCAC-3') and LZT-MA (5'-TAATGGGCTAGGTTACGTTGGTGTAG-3'), and the primer pair LZT-MS (5'-TAACCTAGCCCATTACGGTCAATCC-3') and LZT-D (5'-GGCAACATGGAAATCGC-3') were mixed and used as templates for the second PCR with the primer pair LTZ-U and LZT-D. Replacement of an FspI-AatII fragment of pIK153LZS.6 by the FspI-AatII fragment of the resulting PCR product resulted in pIK153 T10.1. A BamHI-SmaI fragment covering the lacZ gene of LIA7 was inserted into the BamHI site of pIK153 (resulting in pNY19). pNY21 was made by replacing the smaller SmaI-SacI fragment of pNY19 with the homologous SmaI-SacI fragment of pIK153T10.1, which carries the mutant sequence. An XbaI-BglII fragment of pNY21 was used to replace the smaller XbaI-BamHI fragment of pHM5 (resulting in pNY58). pAdNY58 was made by replacement of the smaller I-CeuI-PI-SceI fragment of pAdHM4 with an I-CeuI-PI-SceI fragment of pNY58. The longer PacI fragment of pAdNY58 was transfected into 293 cells. The recombinant adenovirus AdNY58 was prepared and purified from the cell culture.

Figure 3

Design for gene targeting and its detection. (A) The donor carrying the mutant lacZ gene is inserted into an adenovirus vector. The lacZ mutation will be transferred to the lacZ gene of the lambda transgene in the mouse genome. (B) Expected sequence changes and their detection using restriction analysis.

Figure 4

Restriction analysis of the lacZ-negative gene from mice treated with a recombinant adenovirus. (A) AdNY57-injected mouse. The PCR product of the lambda bacteriophage DNA with primers that flank the target site is 288 bp long. The wild-type PCR product is cut with TfiI into 84 and 204 bp fragments, whereas the Glu461Ala mutant PCR product is not cut. Lane M: Marker DNA prepared by HinfI digestion of the plasmid pUC19; 1–12, lacZ-negative bacteriophages from animal number 2; lacZ⁺: Lambda bacteriophage recovered from control mouse; lacZ^-Glu461Gly: lambda bacteriophage LIA15. (B) AdNY58-injected mouse. The PCR product of the lambda bacteriophage DNA with primers that flank the target site is 213 bp long. The Tyr105Stop mutant PCR product is cut with XspI into 146 and 67 bp fragments, whereas the wild-type product is not. Lane M: Marker DNA prepared by HinfI digestion of plasmid pUC19; 1–4, lacZ-negative bacteriophages from animal number 3; lacZ⁺: Lambda bacteriophage recovered from control mouse; lacZ^-Tyr105Stop: lambda bacteriophage LIA11.