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. 2017 Oct 17;8(1):31.
doi: 10.1186/s13293-017-0154-6.

Genetic sex determination of mice by simplex PCR

Simon James Tunster  1
Affiliations

Genetic sex determination of mice by simplex PCR

Simon James Tunster. Biol Sex Differ. .

Abstract

Background: Investigating fetal development in mice necessitates the determination of fetal sex. However, whilst the sex of adult and juvenile mice can be readily distinguished from anogenital distance, the sex of fetal and neonatal mice cannot be identified visually. Instead, genetic sex must be determined by PCR amplification of X chromosome genes with divergent Y chromosome gametologs. Existing simplex PCR methods are confounded by small size differences between amplicons, amplification of unexpected products, and biased amplification of the shorter amplicon.

Results: Primers were designed flanking an 84 bp deletion of the X-linked Rbm31x gene relative to its Y-linked gametolog Rbm31y. A single product was amplified from XX samples, with two products amplified from XY samples. Amplicons were resolved by gel electrophoresis for 20 min, with unbiased amplification of both products observed in XY samples.

Conclusion: This method achieves rapid and unequivocal genetic sex determination of mice in low volume PCR reactions, reducing reagent usage and simultaneously eliminating shortcomings of previous methods.

Keywords: Rbm31x; Rbm31y; Sex genotyping of mice; Simplex PCR.

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Conflict of interest statement

Ethics approval

No animals were generated specifically for this study; only archived yolk sac material was used.

Consent for publication

Not applicable.

Competing interests

The author declares that he has no competing interests.

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Figures

Fig. 1
Fig. 1
Genetic sex determination of mice by amplification of Rbm31y and/or Rbm31x by simplex PCR. a Alignment of Rbm31y (nucleotides 997–1411) and Rbm31x (nucleotides 990–1320). Sequences highlighted in red denote mismatches and the 84 bp deleted region. Primers highlighted in green. Sex determination by simplex PCR of yolk sac lysates from samples of 129S2/SvHsd (b) and C57BL/6JOlaHsd (c) genetic backgrounds. Lane L = PCR Ranger 100 bp Ladder (GeneFlow); Lanes 1–7 = yolk sac lysates; Lane 8 = no template control. d Confirmation of methodology using ear biopsies of adult animals from 129S2/SvHsd (129) and C57BL/6JOlaHsd (BL6) backgrounds. Lane L = PCR Ranger 100 bp Ladder (GeneFlow); Lanes 1–8 = ear biopsy lysates: XY 129, XX 129, XX BL6, XX 129, XY 129, XY BL6, XY 129, XX BL6; Lane 9 = no template control. Arrows indicate 500 bp band
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