Assessment of difference in gene expression profile between embryos of different derivations

Abstract

Researchers have exerted sustained efforts to improve the viability of somatic cell nuclear transfer (SCNT) embryos, testing their experimental designs and probing the resultant embryos. However, the lack of a reliable method to estimate the efficacy of these experimental attempts is a chief hindrance to tackling the low-viability problem in SCNT. Here, we introduce a procedure that assesses the degree of difference in gene expression profiles (GEPs) of blastocysts from each other as a representative control of good quality. We first adapted a multiplex reverse transcription-polymerase chain reaction strategy to obtain GEPs for 15 reprogramming-related genes from single mouse blastocysts. GEPs of individual blastocysts displayed a broad range of variations, the extent of which was calculated using a weighted root mean square deviation (wRMSD). wRMSD-based quantitation of GEP difference (qGEP) found that GEP difference between in vivo-derived blastocysts (in vivo) and SCNT blastocysts was greater than the difference between in vivo blastocysts and in vitro-produced (IVP) blastocysts, demonstrating that the SCNT group was more distantly related to the in vivo group than the IVP group. Our qGEP approach for grading individual blastocysts would be useful for selecting a better protocol to derive embryos of better quality prior to field applications.

FIG. 1.

Multiplex reverse transcription-polymerase chain reaction (RT-PCR) of 15 reprogramming-related genes in mouse blastocysts. (A) Multiplex RT-PCR strategy. One-step RT-PCR was used as the first round of PCR (PCR-1); the products were advanced to the second-round, nested PCR (PCR-2). (B) A representative gel image (top), and the relative positions of PCR products of individual genes (bottom). (C) Intensity profiles of multiple PCR bands. Each peak with a different height and size is denoted with gene symbol. The AxioVision intensity profile tool (v. 4.8) was used to measure the band density of PCR products resolved in each lane. (D) Control experiments for multiplex RT-PCR using total RNA from J1 mouse embryonic stem cells. Gel images show multiplex RT-PCR results obtained from omitting the primer sets for the genes of each category in PCR-1. No significant difference is shown in the overall pattern and quantity of the final (PCR-2) products of the other three groups that are boxed in dashed lines, indicating no severe cross-interference of the multiple sets of primers during multiplex RT-PCR. (Square, round, asterisk, and triangle) Expression levels of individual genes in DMs-, HTMs-, PcGs-, and HDMs-negative PCR setup, respectively. (E) Different expression profiles, or expression signatures, of reprogramming-related genes in different types of cells in spline-type plots. Each profile was obtained from four to five replications of multiplex RT-PCR using total RNA. In the case of mouse oocytes, multiplex RT-PCR was performed four times with single mature oocytes. Because of the limited amount of material, the oocytes show the largest variance in expression levels among the cell samples. Error bars represent the standard deviation. mEF, mouse embryonic fibroblast. DMs, DNA methyltransferases (Dnmt1, Dnmt3a, and Dnmt3b); HMTs, histone H3K9-specific methyltransferases (Glp, Setdb1, G9a, and Suv39h1); PcGs, Polycomb-group proteins (Yy1, Suz12, Eed, and Bmi1); and HDMs, histone demethylases (Lsd1, Jhdm3a, Jhdm2a, and Jmjd3).

FIG. 2.

Gene expression profiles of 15 reprogramming-related genes in single mouse blastocysts. (A) Representative density profiles of gene expressions. Each panel represents a density profile of a single blastocyst (10 profiles per each group) and the inset shows the corresponding gel image with lanes of individual gene groups (see Fig. 1B). In vivo–derived, IVP, and SCNT-derived blastocysts were used as templates in multiplex RT-PCR. To avoid complexity, only the HMTs (blue) and HDMs (red) gene peaks are presented. The gene responsible for each peak is denoted in the first panel of the IVP group. The AxioVision intensity profile tool (v. 4.8) was used. (B and C) GEPs of single blastocysts in each blastocyst group (B) and the mean GEPs of individual blastocyst groups (C). In the spline-type plots in B, each line with a different color denotes the expression profile of the 15 genes in a single blastocyst. The number of blastocysts examined is indicated in the parentheses. The Lsd1 level was set to 1.0 as the normalization standard. Error bars, standard error.

FIG. 3.

Quantitative analysis of GEP difference in individuals of the same blastocyst group. (A) GEPs of representative blastocysts with the lowest (min wRMSD) and the highest wRMSD (max wRMSD) are shown along with the mean GEP for each group. (B) The distribution of wRMSDs of individual blastocysts in each group. The deviation of each blastocyst's GEP from the mean of each group was estimated using a wRMSD. Samples are sorted in order by wRMSD value. The numbers in parentheses indicate the mean wRMSD±standard deviation, which is marked as a dashed line. The closer the wRMSD is to zero, the closer the GEP is to the mean GEP of its own group.

FIG. 4.

Difference in the GEPs of IVP and SCNT blastocysts compared to the in vivo blastocysts. (A) Relative wRMSD distribution. wRMSDs were obtained by calculating the deviations of the gene expression in each IVP blastocyst (IVP/In vivo) and SCNT blastocyst (SCNT/In vivo) from the mean GEP of the in vivo group (see Fig. 3B). Samples are sorted in order by wRMSD value. The dashed line in each panel indicates the mean wRMSD of the in vivo group as the reference (see Fig. 3B). (B) Proportion of blastocysts that belong to different wRMSD ranges. (C) Statistics for wRMSD values between in vivo and IVP (left) and between in vivo and SCNT (right) blastocysts. The p values between different blastocyst groups are indicated (t-test). The relative wRMSDs of individual blastocysts are presented as crosses on the left of each box plot. The mean values are indicated in the boxes (circles). Box whiskers indicate the range from 5% to 95%.