Quick sex determination of mouse fetuses
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.
References (14)
- et al.
Basic local alignment search tool
J. Mol. Biol.
(1990) - et al.
Genomic DNA from mice: a comparison of recovery methods and tissue sources
Biochem. Mol. Med.
(1997) - et al.
Transfer of the human glucocerebrosidase gene into hematopoietic stem cells of nonablated recipients: successful engraftment and long-term expression of the transgene
Blood
(1995) - et al.
Generation of five high-complexity painting probe libraries from flow-sorted mouse chromosomes
Genomics
(1994) - et al.
Growth of a rat neuroblastoma cell line in serum-free supplemented medium
Proc. Natl. Acad. Sci. USA
(1979) - et al.
Rapid sexing of murine preimplantation embryos using a nested, multiplex polymerase chain reaction (PCR)
Mol. Reprod. Dev.
(1998) - et al.
Inverted repeat structure of the Sry locus in mice
Proc. Natl. Acad. Sci. USA
(1992)
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