266 WHOLE GENOME AMPLIFICATION ON BLASTOMERS OF POST-BIOPSY BOVINE EMBRYO

Abstract

Biopsy of embryos is very useful for choosing the desired sex and for production of cloned and transgenic livestock. However, only a small amount of genomic DNA is available to perform genetic studies. Alternatively, methodologies using whole genome amplification (WGA) have been developed. The aims of this study were to evaluate the effect of WGA on blastomeres removed from 8- to 16-cell bovine embryos and to determine the sex of blastomeres. Oocytes obtained from slaughterhouse ovaries were in vitro matured and fertilized. On the fourth day after fertilization, 8- to 16-cell bovine embryos were biopsied, and one-fourth of an embryo was removed. The blastomeres (n = 56) were submitted to WGA followed by PCR. Prior to the whole genome amplification, male and female bovine DNA samples were serially diluted (30 ng μL^–1, 3.0 ng μL^–1, 0.3 ng μL^–1, 0.03 ng μL^–1, 0.003 ng μL^–1, 0.0003 ng μL^–1) and embryos of various development stages (2, n = 6; 4–7, n = 5; ≥8-cell, n = 5; blastocyst n = 27) were used to standardize PCR protocols and set the amplification limits. To digest the cellular cytoplasm and release the genomic DNA, embryos and blastomeres were submitted a 3 mg mL^–1 proteinase K before PCR. Next, blastomeres were submitted to the GenomiPhi DNA Amplification Kit (GE Healthcare) according to manufacturer’s instructions. The product (1 μL) was electrophoresed on a 1% agarose gel stained with 3.0 μg mL^–1 ethidium bromide. The reaction mixture was added to the material to be amplified (2 mm MgCl2, 5X PCR buffer, 0.2 mm each dNTPs, 0.05 U μL^–1 GoTaq DNA polymerase, 0.25 μm of primer). The products were submitted to electrophoresis on 8% polyacrilamide gel and stained with silver nitrate procedure. The chi-square test was used for statistic evaluation of the results to test the WGA efficiency and to determine the sex rates of bovine embryos and biopsied samples submitted to PCR. It was possible to achieve 98% efficiency in amplifying blastomeres using the WGA kit. Amplified samples showed approximately 400 ng of DNA generated from an estimated initial amount of 12 pg of DNA resulting from two cells per embryo. In whole embryos from different stages, no diffference was detected in the proportion of sexes (P > 0.05). However, a greater number of female samples was noted in biopsied material (76%, 25/33) (P < 0.05). PCR efficiency in blastocysts (93%, 2/27) was statistically greater (P < 0.05) than embryos in early stages of development (83%, 5/6), and biopsied material to 2, 4–7 and ≥8-cell (40%, 2/5; 60%, 3/5; and 59%, 33/56; respectively). These differences could be related to a sex-chromosomal mosaicism or absence of a nucleus in biopsied samples. The WGA creates a DNA stock sample that could be used for various gene profiling and sex determination analyses.

Financial support: Fapemig, CNPq.