Mouse mutants from chemically mutagenized embryonic stem cells

Abstract

The drive to characterize functions of human genes on a global scale has stimulated interest in large-scale generation of mouse mutants. Conventional germ-cell mutagenesis with N-ethyl-N-nitrosourea (ENU) is compromised by an inability to monitor mutation efficiency, strain and interlocus variation in mutation induction, and extensive husbandry requirements. To overcome these obstacles and develop new methods for generating mouse mutants, we devised protocols to generate germline chimaeric mice from embryonic stem (ES) cells heavily mutagenized with ethylmethanesulphonate (EMS). Germline chimaeras were derived from cultures that underwent a mutation rate of up to 1 in 1,200 at the Hprt locus (encoding hypoxanthine guanine phosphoribosyl transferase). The spectrum of mutations induced by EMS and the frameshift mutagen ICR191 was consistent with that observed in other mammalian cells. Chimaeras derived from ES cells treated with EMS transmitted mutations affecting several processes, including limb development, hair growth, hearing and gametogenesis. This technology affords several advantages over traditional mutagenesis, including the ability to conduct shortened breeding schemes and to screen for mutant phenotypes directly in ES cells or their differentiated derivatives.

Fig. 1

Breeding strategy for recovery of recessive mutations. Chimaeras generated from EMS-treated ES cells were mated with C57BL/6J (B6) partners. Agouti G1 progeny derived from gametes of ES cell origin were mated with either opposite sex G1 siblings (left) or B6 (right). We backcrossed G2 progeny with the opposite sex G1 parent to yield the G3 generation. Because each of the founding chimaeras could transmit two different alleles at each locus (assuming a germ line derived from a single founder ES cell), several G1 progeny from each chimaera were used to initiate these screens. Furthermore, to optimize the likelihood that most mutagenized alleles would be captured in this screen, we sought to generate 6–8 G2 progeny from each G1 male (up to four G2s from a female G1) for backcrossing.

Fig. 2

Phenotypes of mutants. a, Bone preparations of hindfeet from normal (+/+, left) and Pde (+/−) mutant mice. The length of the extra toe can vary, having either two phalanges, as is seen in the normal first digit, or three phalanges, as in digits two through five. In some cases, the extra toe arises as a bifurcation of a digit 1 metacarpal or phalange. Arrows highlight the variable manifestation of the poly-dactyly phenotype in hindfeet of one individual. L, left; R, right. b, Examples of sne phenotypic expression in hindfeet of homozygous mutants. c, Light microscopic and electron microscopic (EM; inset) images of sperm from the rsph mutant and associated sgdp phenotype. Normal sperm heads in mice are hook-shaped; an apparently normal shaped head is enveloped in the residual cytoplasmic sac in the EM photograph. Right, an example of an sgdp testis section in which relatively normal (albeit with apparently abnormal flagellar development in spermatids) seminiferous tubule segments are juxtaposed with agametic tubule segments. d, Skeletal preparations of the twl mouse. Arrows indicate the malformed fibulae in the mutant (−/−) compared with a normal animal (+/+). Also note the shortened and thickened tibiae in the affected mice. e, A pfz homozygote. f, Cross-section through the testes of a mei1 mutant and a normal animal (+/+). Note the accumulation of spermatocytes with condensed chromosomes in the most mature mutant tubules, and how others are either vacuolated or lined only by a thin spermatogonial layer. Presumed apoptotic cells appear as large, dark cells. Scale bars in c,f, 100 μM.