Single-step PCR-based genetic sex determination of rat tissues and cells

Abstract

The advent of genome editing strategies has expanded the range of animal models available for gene manipulation and renewed research interest in the rat. Gender is a key variable for in vivo gene function analyses. Here, we present a simple PCR-based method to determine genetic sex in the rat.

Keywords: fetal; rat; sex determination.

Fig. 1. Locations of primers for Kdm5c (X chromosome, NC_005120.4) and Kdm5d (Y chromosome, NC_024475.1) genes used in rat sex determination

A 19-nucleotide reverse primer (R) for Kdm5c has a single nucleotide mismatch (underlined) for Kdm5d at nucleotide 532649 of the Y chromosome. Unique forward primers F1 and F2 correspond to Kdm5c on the X chromosome and Kdm5d on the Y chromosome, respectively.

Fig. 2. Distinct PCR products corresponding to X and Y chromosome-specific genes

Panel A: L) 100-bp plus DNA ladder; 1) male trophoblast stem (TS) cells; 2) female TS cells; 3) gestation day (gd) 14.5 male fetus; 4) gd 14.5 female fetus; 5) gd 18.5 male fetus; 6) gd 18.5 female fetus; 7) adult male Holtzman Sprague Dawley (HSD) rat; 8) adult female HSD rat; 9) adult male Brown Norway (BN) rat; 10) adult female BN rat. Panel B: L) 100-bp plus DNA ladder; 1) PC-12 (adrenal medulla); 2) MMQ (pituitary); 3) Rcho-1 (trophoblast); 4) NR8383 (lung macrophage); 5) NB2 (T cell); 6) RNK16 (natural killer cell); 7) R2C (Leydig cell); 8) adult female Holtzman Sprague Dawley (HSD) rat; 9) adult male HSD rat.