The chloramphenicol acetyltransferase gene under the control of the late E2A promoter of adenovirus type 2 (Ad2) was introduced as transgene into the B6D2F1 mouse strain with mixed genetic background and became extensively de novo methylated. The methylation of this pAd2E2AL-CAT (7-1A) transgene was regulated in a strain-specific manner apparently depending on the site of integration. Transmission of the 7-1A transgene into an inbred DBA/2, 129/sv, or FVB/N genetic background led to a significant loss of methylation in the transgene, whereas C57BL/6, CB20, and Balb/c backgrounds favored the de novo methylation in very specific patterns. The newly established patterns of de novo methylation were transmitted to the offspring and remained stable for many generations, regardless of the heterozygosity of strain-specific DNA sequences present in these mouse strains. Segregation analyses showed a non-mendelian transmission of methylation phenotypes and suggested the involvement of dominant modifiers of methylation. The genotype-specific modifications of the transgene were followed for 11 backcross generations. These observations reflect an evolutionarily conserved mechanism directed against foreign, e.g. viral or bacterial, DNA at least in the chromosomal location of the 7-1A transgene. In seven additional mouse lines carrying the same transgene in different chromosomal locations, strain-specific alterations of methylation patterns were not observed.
The methylation status of a transgene, which carried the adenovirus type 2 E2A late promoter linked to the chloramphenicol acetyltransferase gene, was studied in three transgenic mouse lines (5-8, 7-1 and 8-1). These lines were analysed over a large number of offspring generations beyond the founder animal. In mating experiments, the influence of the parent-of-origin and strain-specific backgrounds on the transgene methylation patterns were assessed and found to have no effect on the pre-established methylation patterns in mouse lines 5-8 and 8-1. The founder animal 7-1 carried two groups of a total of ten transgenes, which were located on two different chromosomes. These arrays of transgenes could be segregated into separate mouse lines 7-1A and 7-1B. The transgenes of 7-1A animals exhibited cellular mosaic methylation patterns that were demethylated in approximately 10% of the offspring in a mixed genetic background. Upon further transmission of these transgenes in a mixed genetic background, the grandparental methylation patterns were reestablished in most progeny. Mating to inbred DBA/2 mice resulted in maintenance of the demethylated pattern or in further demethylation of the transgenes in approximately 50% of the offspring. In contrast, an equal number of transgenic siblings from matings to C57BL/6 mice showed a return to the original methylation pattern. The mosaic methylation status of this locus was apparently controlled by mouse-strain-specific factors. The methylation patterns of the 7-1B transgenes were not cellular mosaic and remained stable in all offspring, as with lines 5-8 and 8-1. Hence, the strain-dependent and cellular mosaic transgene methylation patterns of 7-1A animals were probably a consequence of the chromosomal integration site of the transgenes (position effect).
Pipetting and concentration measurement of viscous ultra-high-molecular-weight (UHMW) DNA samples is challenging and often highly imprecise. Effective guidelines for handling UHMW samples are missing in the field. Herein, a simple and low-cost workflow is presented that enables accurate pipetting and reliable concentration measurement. Central to the workflow is the shearing of representative small aliquots of UHMW DNA samples to a fragment size <150 kb by vortexing them for 1 min with a glass bead in a round-bottomed 2-ml tube. Additionally, a solution is provided for accurate quantitation of high-molecular-weight DNA with fluorometric (Qubit [Thermo Fisher Scientific, MA, USA]) methods by using an appropriate genomic DNA standard, resulting in values that match spectrophotometric (Nanodrop [Thermo Fisher Scientific]) optical density readings.
In previous work from this laboratory, an inverse dependence was established for the adenovirus type 2 E2A late promoter between sequence-specific DNA methylation and promoter activity [1-5; for reviews see ref. 6, 7]. The effect of DNA methylation on promoter activity was also assessed in the transgenic mice, which were obtained from microinjections of unmethylated or in vitro HpaII-premethylated pAd2E2AL-CAT DNA [1] into F2 zygotes from B6D2F, (C57BL/6 × DBA/2) hybrid mice. In CAT assays carried out on organ extracts from the pAd2E2AL-CAT mice, the inverse relationship was confirmed [2].We studied the stability of the pAd2E2AL-CAT DNA methylation patterns in up to eight mouse generations and assessed the influence of the strain-specific genetic background. Three pAd2E2AL-CAT mouse lines were crossed with inbred DBA/2, C57BL/6 or B6D2F, mice. Parent-of-origin effects were controlled by exclusive hemizygous transgene transmission either via females or males. The founder animal of line 7-1 carried two groups of transgenes (A and B) on separate chromosomes. The transgene methylation patterns of the 7-1B transgenes and those of the lines 5-8 and 8-1 were stably transmitted.Southern blot hybridization experiments [8, 9] revealed that the 7-1A transgene methylation pattern was a cellular mosaic. In mixed-genetic-background offspring from 7-1A animals, 10% carried transgenes with HpaII-DNA methylation levels that were reduced from 40 to 10-15%. This finding suggested that in this background the factors that supported high methylation levels were dominant. When inbred DBA/2 mice were the mates, 40% of the siblings carried demethylated transgenes, whereas this ratio amounted to only 10% in C57BL/6 offspring (comparable to B6D2F1 crossings). Transgene methylation patterns were not detectably influenced by the parent-of-origin.
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