Elsevier

Journal of Neuroscience Methods

Volume 95, Issue 2, 15 February 2000, Pages 127-132
Journal of Neuroscience Methods

Quick sex determination of mouse fetuses

https://doi.org/10.1016/S0165-0270(99)00157-0Get rights and content

Abstract

We designed a rapid, simple and accurate PCR method to determine sexual identity of mouse fetuses collected on embryonic day 15. A multiplex PCR amplification was used to detect male-specific sequence (Sry) in DNA extracted from fetal livers through SDS denaturation followed by high salt extraction and precipitation. This extraction method resulted in sufficiently purified DNA in <1 h and was suitable for PCR. The DNA obtained was amplified using a robot thermal cycler for 33 cycles. The reaction was performed in 50 μl, using two sets of primers specific for Sry gene (chromosome Y) and IL3 gene (chromosome 11). Amplification duration was 1.5 h. The assessment of the results was done by electrophoresis in 3% agarose run at high voltage. The 402 bp band (Sry) obtained identifies the male fetuses and the 544 bp product (IL3) confirms the correct amplification of the template DNA. The entire procedure took <4 h. The specificity of the method was confirmed by fluorescent in situ hybridization using a specific male probe on cultured male and female neural stem cells. This method allowed the preparation and culture of pure male and female neural stem cells from fetal tissue.

Introduction

Several polymerase chain reaction (PCR) methods have been described for sexing mouse embryos (Han et al., 1993, Greenlee et al., 1998). These methods were optimized for sexing of single cells from murine embryos by nested PCR and require a full day of work before producing a result. When cultivating materials from fetuses, such as neural stem cells, individual fetuses need to be pooled. In order to prepare sex-specific cultures, it is necessary to develop a faster procedure for sexual determination.

Most sexing methods use DNA extracted by proteinase K digestion followed by organic extraction and DNA precipitation. This extraction procedure requires at least 3 h. A rapid DNA extraction method using high salt protein extraction followed by ethanol precipitation has been previously optimized (Lahiri and Schnabel, 1993, Hofstetter et al., 1997). This method allowed the preparation of purified DNA from blood samples suitable for molecular biology. By modifying their method, we were able to extract embryonic liver DNA in <1 h.

PCR detection of Y chromosome allows rapid and accurate identification of male DNA. Sex determining region protein gene (Sry) (Koopman et al., 1991, Gubbay et al., 1992) is encoded by Y chromosome and well conserved among species. However, PCR amplification is typically performed with thermal cyclers, and usually requires up to 2.5 h including heating and cooling periods. The amplification time required for PCR can be reduced by the use of a robot thermal cycler containing several heating blocks that can be programmed at different temperatures, therefore eliminating the heating and cooling periods.

We combined the use of high salt DNA extraction, a robot thermal cycler and a rapid electrophoresis to design a fast protocol for the amplification of sex-specific DNA. This new method allowed reliable sexing of fetuses in <4 h, and permitted sexing fetuses before processing tissues. In this report, we utilize this rapid sexing method to obtain EGF-dependent striatal neural precursor cells from exclusively female or male embryonic day 15 brains.

Section snippets

Source of fetuses

Mice (BALB/c males and females) were purchased from Taconic Farms (Germantown, NY). The animals were bred and the females were checked for vaginal plugs daily to establish day 1 of gestation. Pregnant females were group-housed (3–4 per cage). All animals received food and water ad libitum. On embryonic day 15, pregnant females were sacrificed by cervical dislocation and fetuses dissected and conserved on Petri dishes (Falcon, Becton-Dickinson, Franklin Lakes, NJ) filled with culture medium (see

DNA extraction

The DNA extraction using high salt protein precipitation gave good quality DNA with minimal RNA contamination and complete protein decontamination. In order to evaluate average DNA quality, 27 samples were analyzed by spectrophotometer. The ratio of UV absorbency at 260–280 nm averaged 2.00±0.01 and the concentration of dissolved DNA averaged 339±29 mg/ml. The range of DNA concentration was 157–769 mg/ml. Electrophoresis evaluation of the size of the genomic DNA obtained showed an average size

Discussion

This PCR technique was developed to rapidly and accurately determine the sex of mouse fetuses. Its originality resides in the fact that all steps of the PCR procedure were optimized with regards to timing in order to perform it in <4 h. As compared with other sexing methods, it is faster by at least 2 h (Han et al., 1993, Greenlee et al., 1998). The gains were made at several steps: DNA extraction, PCR and electrophoresis. The extraction of liver DNA using SDS denaturation followed by high salt

Acknowledgements

This work was supported by a NIH grant no. PO1 CA68426 and an Advanced Research Fellowship from the Swiss National Science Foundation (J.-F. Lambert). We are grateful to Dr Todd Savarese for insightful comments on the manuscript.

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