Mutant mice derived by ICSI of evaporatively dried spermatozoa exhibit expected phenotype

Abstract

Apolipoprotein E (Apoe)-deficient knockout mice were used to test the hypothesis that mutant mice preserved as evaporatively dried (ED) spermatozoa, stored at -80 °C for 6 months, and then recovered by ICSI will exhibit the same phenotype as before preservation. The birth rate of mice recovered by ICSI of evaporatively dried spermatozoa was lower than that of fresh spermatozoa (17.5 vs 38.0%). Progeny of mice preserved using evaporatively dried spermatozoa were reproductively sound. From these, the second generation of mice produced by natural mating showed lesions typical of APOE deficiency, including severe hypercholesterolemia, hypertriglyceridemia, markedly increased plasma low-density lipoprotein level, and extensive and severe atherosclerotic lesions in the aorta. We conclude that the expected phenotype caused by an induced genetic mutation can be faithfully recapitulated and sustained in subsequent generations of mice preserved and stored as ED spermatozoa and recovered using ICSI. Because it is simpler, faster, and cheaper than conventional (cryopreservation) and nonconventional (freeze-drying) preservation procedures, evaporative drying is a viable, cost-effective, and efficient method for preserving and storing valuable mutant mouse strains.

Figure 1

The plasma totalcholesterol levels ofthe second-generation mice in the fresh and evaporatively dried (ED) Apoe^−/− spermatozoon groups compared with that of the control group mice. *P<0.0001 compared with that of fresh spermatozoon group or ED spermatozoon group.

Figure 2

The plasma triglyceride levels of the second-generation mice in the fresh and evaporatively dried (ED) Apoe^−/− spermatozoon groups compared with that of the control group mice. *P<0.001 compared with that of fresh spermatozoon group or ED spermatozoon group.

Figure 3

The plasma low-density lipoprotein (LDL) levels of the second-generation mice in the fresh and evaporatively dried (ED) Apoe^−/− spermatozoon groups compared with that of the control group mice. *P<0.0001 compared with that of fresh spermatozoon group or ED spermatozoon group.