94 FEASIBILITY OF WHOLE-GENOME AMPLIFICATION OF LYSED CELLS FROM EMBRYO BIOPSIES FOR PRE-IMPLANTATION GENETIC DIAGNOSIS

Abstract

Pre-implantation genetic diagnosis has created the ability for embryos to be screened before transfer into the uterus, but there is only a very small quantity of genomic DNA in biopsy material from early embryos. Using a whole-genome amplification (WGA) procedure in conjunction with marker-assisted selection provides the ability to scan the entire genome and screen multiple genetic traits, allowing producers to determine the genetic value of the embryos and select ones with high performing traits. Current WGA protocols use purified DNA, but it is impractical to do DNA purification on an embryo biopsy and typical lysis buffers contain sodium dodecyl sulfate, which is inhibitory to amplification. The objective of this study was to use WGA and PCR to determine the feasibility of amplifying target genes in lysed bovine fibroblast cells at a level appropriate from embryo biopsies for pre-implantation genetic diagnosis. To ensure that WGA is representative of the entire genome and that there is no bias present, bovine fibroblast cells were first tested to determine the consistency and viability of amplification without DNA purification from a cell number appropriate for cell biopsy. Fibroblast cells were cultured in DMEM with 10% fetal bovine serum at a dilution level of ~1000 cells, and then aliquots of a single cell suspension of 2 to 10 cells were created to represent biopsy levels. Five target genes were tested: adiponectin, calpain, calpastatin, growth hormone 1, and leptin. Using cultured cells, it was determined that a crude lysis procedure using boiling water, in addition to performing single cell amplification, and nested PCR were most successful in amplifying the target genes. Two to four aliquots from 6 different lysed bovine embryos were tested for proper amplification using REPLI-g Single Cell Whole Genome Amplification Kit (Qiagen, Valencia, CA, USA) followed by nested PCR using primers of target genes created via GenBank. The DNA was quantified using a spectrophotometer before and after amplification. Originally, the starting DNA was diluted to a median of 4.0 ng μL⁻¹, yielding an average of 466.6 ± 61.4 ng μL⁻¹. To increase the yield, the DNA was then diluted to 9.5 ng μL⁻¹. After performing WGA, the yield was 3047.4 ± 191.4 ng μL⁻¹, amplifying the DNA at a rate of ~16 μg per 50 μL reaction. To determine whether amplification of the target genes was successful, PCR material was run on a 2% agarose gel using gel electrophoresis and results were analysed under UV light using ChemiDoc XRS (Bio-Rad, Hercules, CA, USA) image analysis. Eleven out of 16 aliquots (68.75%) successfully amplified all 5 target genes. The remaining 5 aliquots resulted in partial amplification, amplifying either 3 or 4 out of the 5 target genes. In conclusion, using a whole genome amplification procedure in conjunction with crude lysis and nested PCR on embryos provides a sufficient quantity of genomic DNA for use in amplification of target genes for pre-implantation genetic diagnosis to determine improved productivity in the calf before implantation.