Transgenic λ medaka as a new model for germ cell mutagenesis

Abstract

To address the need for improved approaches to study mutations transmitted to progeny from mutagen-exposed parents, we evaluated lambda transgenic medaka, a small fish that carries the cII mutation target gene, as a new model for germ cell mutagenesis. Mutations in the cII gene in progeny derived from ethyl-nitrosourea (ENU)-exposed males were readily detected. Frequencies of mutant offspring, proportions of mosaic or whole body mutant offspring, and mutational spectra differed according to germ cell stage exposed to ENU. Postmeiotic germ cells (spermatozoa/late spermatids) generated a higher frequency of mutant offspring (11%) compared to premeiotic germ cells (3.5%). Individuals with cII mutant frequencies (MF) elevated more than threefold above the spontaneous MF (3 x 10(-5)) in the range of 10(-4) to 10(-3) were mosaic mutant offspring, whereas those with MFs approaching 1 x 10(-2) were whole body mutant offspring. Mosaic mutant offspring comprised the majority of mutant offspring derived from postmeiotic germ cells, and unexpectedly, from spermatogonial stem cells. Mutational spectra comprised of two different mutations, but at identical sites were unusual and characteristic of delayed mutations, in which fixation of a second mutation was delayed following fertilization. Delayed mutations and prevalence of mosaic mutant offspring add to growing evidence that implicates germ cells in mediating processes postfertilization that contribute to genomic instability in progeny. This model provides an efficient and sensitive approach to assess germ cell mutations, expands opportunities to increase understanding of fundamental mechanisms of mutagenesis, and provides a means for improved assessment of potential genetic health risks.

Subchronic perfluorooctanesulfonate (PFOS) exposure induces elevated mutant frequency in an in vivo λ transgenic medaka mutation assay

“…However, the majority of frameshifts were +1 insertions at thiamine and adenine in the 1,1-DCP treated fish while the majority of frameshifts in PFOS treated fish were +1 insertions at guanine in the CpG sites. In contrast, the spontaneous frameshift mutations consist primarily of −1 frameshift deletions of guanines in the string of repetitive guanines or at CpG sites2326, which was also evidenced in the control fish of the present study. However, significant increase of −1 frameshift mutations involving the loss of G:C base pairs has also been reported in the prostate of Big Blue ® transgenic rats exposed to the potent mutagen of 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine36.…”

“…Transgenic mutation assays, unlike traditional mutation analysis in vivo , are sensitive, not limited to any particular tissues, and permit the screening of a large number of copies of a locus quickly232425262728. The λ transgenic medaka, a transgenic fish model for in vivo mutation analysis, carries multiple copies of the λ bacteriophage vector that harbors the cII gene as a mutant target23.…”

Isolated spermatozoa as indicators of mutations transmitted to progeny

“…1). The cII MFs in isolated spermatozoa collected from each male prior to ENU treatment (male A,1.2 × 10 −5 , and male B, 0.9 × 10 −5 ), and in spermatozoa collected 5 days following mutagen treatment were not significantly different from one another (male A, 1.7 × 10 −5 , and male B, 1.1 × 10 −5 ), or from historical spontaneous cII MFs (2.5 × 10 −5 ) [25]. Similarly, cII MFs in progeny derived from spermatozoa collected from each male prior to ENU exposure (0.9 to 4.8 × 10 −5 ) were not significantly elevated (Fig.…”

“…Genomic DNA was isolated from spermatozoa or from individual progeny using procedures described previously [22,25,28]. Briefly, spermatozoa or whole animals were digested with proteinase (1 × SSC, 20% SDS, 20 mg/ml proteinase K) and extracted with equal volumes of phenol: chloroform.…”

“…Recently, the λ transgenic medaka, a small fish mutation model shown previously to be a sensitive and reliable model for detecting mutations in somatic cells [18–24], was introduced as an alternative to traditional mouse models used to detect mutations in progeny derived from mutagen-exposed male germ cells [25]. In addition to providing numerous practical benefits, such as prolific reproductive capacity, breeding with multiple untreated females over consecutive days prior to and after mutagen exposure, and ability to analyze mutations using individuals or pools of offspring, the study strengthened growing evidence of common processes of DNA damage and repair shared among distantly related species.…”