Conserving, Distributing and Managing Genetically Modified Mouse Lines by Sperm Cryopreservation

G Charles Ostermeier,Jane S Farley,Robert A Taft,Michael V Wiles,Hany A El-Shemy

doi:10.1371/journal.pone.0002792

Abstract

BackgroundSperm from C57BL/6 mice are difficult to cryopreserve and recover. Yet, the majority of genetically modified (GM) lines are maintained on this genetic background.Methodology/Principal FindingsReported here is the development of an easily implemented method that consistently yields fertilization rates of 70±5% with this strain. This six-fold increase is achieved by collecting sperm from the vas deferens and epididymis into a cryoprotective medium of 18% raffinose (w/v), 3% skim milk (w/v) and 477 µM monothioglycerol. The sperm suspension is loaded into 0.25 mL French straws and cooled at 37±1°C/min before being plunged and then stored in LN2. Subsequent to storage, the sperm are warmed at 2,232±162°C/min and incubated in in vitro fertilization media for an hour prior to the addition of oocyte cumulus masses from superovulated females. Sperm from 735 GM mouse lines on 12 common genetic backgrounds including C57BL/6J, BALB/cJ, 129S1/SvImJ, FVB/NJ and NOD/ShiLtJ were cryopreserved and recovered. C57BL/6J and BALB/cByJ fertilization rates, using frozen sperm, were slightly reduced compared to rates involving fresh sperm; fertilization rates using fresh or frozen sperm were equivalent in all other lines. Developmental capacity of embryos produced using cryopreserved sperm was equivalent, or superior to, cryopreserved IVF-derived embryos.Conclusions/SignificanceCombined, these results demonstrate the broad applicability of our approach as an economical and efficient option for archiving and distributing mice.

Highlights

Embryo cryopreservation is an effective strategy for managing mouse lines
Conclusions/Significance: Combined, these results demonstrate the broad applicability of our approach as an economical and efficient option for archiving and distributing mice
In our experience (Table 1) and in that of others[2,3,4,5], the impaired fertility associated with cryopreserved mouse sperm is dependent on genetic background, with sperm from the C57BL/6 backgrounds being sensitive

Summary

Introduction

Its adoption has been limited by the cost, time and the number of animals required This is especially true for those lines where embryo yields are low, e.g. BALB/c. In our experience (Table 1) and in that of others[2,3,4,5], the impaired fertility associated with cryopreserved mouse sperm is dependent on genetic background, with sperm from the C57BL/6 backgrounds being sensitive. This strain is one of the most commonly used for creating and maintaining genetically modified (GM) lines. The majority of genetically modified (GM) lines are maintained on this genetic background

Objectives

Methods

Results