Functional expression study of igf2 antisense transcript in mouse

Abstract

Insulin-like growth factor antisense gene (Igf2as) expression was investigated in different mouse tissues during development, in differentiating C2C12 cells and in a ΔDMR1-U2 knockout mouse model. The expression levels of Igf2as were high in fetal and newborn liver and muscle tissues compared to adults. The Igf2as gene was also expressed in placenta and in brain. The expression data suggests that the Igf2as gene plays a role in early development of the mouse and in placenta. There was no consistent evidence for an interaction between Igf2 and Igf2as transcripts. Furthermore, in knockout placentas lacking Igf2as transcription, Igf2 expression was comparable to that in wild type. These results indicate that Igf2as does not regulate Igf2 sense transcripts. In previous studies, it was suggested that the ΔDMR1-U2 knockout mouse showing intrauterine growth restriction was caused by the absence of placenta-specific Igf2 P0 transcription. We conclude that the ΔDMR1-U2 deletion phenotype should be reconsidered in the light of a functional Igf2as gene.

Figure 1

Structure of Igf2 gene, Igf2as, and ΔDMR1-U2 mutation. The arrows indicate the five promoters (P0, Pm, P1, P2, and P3) for Igf2 and(P) for Igf2as. DMR1 indicates the position of the DMR1 regulatory region. V1, V2, V3, P0, and Pm indicate the five variants of Igf2 gene. The 5 kb deleted region in ΔDMR1-U2 mice is indicated.

Figure 2

Quantification of expression levels of Igf2 variants and Igf2as transcripts in different tissues. Igf2 variants and Igf2as were quantified in muscle (a), liver (b), and brain (c) tissues of fetus, newborn (NB), and adult and included embryonic brain. V1, V2, V3, and Igf2as stand for Igf2 variants 1, 2, 3, and Igf2 antisense, respectively. The results are expressed in relative expression, calculated relative to the first value. Each bar represents the mean of three samples each analyzed in triplicate. The standard deviations are indicated as error bars. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 3

Quantification of Igf2 and Igf2as expression during C2C12 myoblast differentiation. The C2C12 cells were maintained in differentiation media (DM) during a total time of 144 h. In the graph V3 and Igf2as correspond to Igf2 variant 3 and Igf2as transcripts, respectively. The results are presented in relative expression. Each bar represents the mean of cells from three culture flasks each quantified in triplicate. The standard deviation for each time point is indicated. The images of C2C12 murine myoblast cells correspond to (a) C2C12 in growth media, (b) C2C12 after 6 h, (c) 24 h, (d) 48 h, (e) 96 h, and (f) 144 h in DM. The scale bar is indicated (30 μm). *P < 0.05 and ***P < 0.001.

Figure 4

Quantification of Igf2 variants expression levels in wild-type and ΔDMR1-U2 knockout placenta. Quantification of Igf2 transcripts performed in wild-type (WT) and knockout (KO) placentas at different embryonic stages (E12, E14, E16, and E19). Igf2 variants 1 (a), 2 (b), and 3 (c) were measured by qPCR. The results are expressed in relative expression. Each bar represents the mean of three samples each quantified in triplicate. The standard deviation is indicated by error bars. *P < 0.05.

Figure 5

Quantification of Igf2 P0 and Igf2as expression levels in wild-type placentas. Quantifications of Igf2 P0 (P0) and Igf2as transcripts levels in wild-type placenta (WT) at different embryonic stages (E12, E14, E16, and E19). The results are expressed in relative expression. Each bar represents the mean of three samples analyzed in triplicate. The standard deviation for each bar is indicated by error bars. *P < 0.05.