*2.1. LIN28 Knockdown in Trophectoderm Resulted in Reduced Proliferation of Trophoblast Cells and Lower Expression of Proliferation-Associated Genes*

Day 9-hatched blastocysts were infected with lentiviral particles expressing shRNA to knockdown LIN28A (AKD) or LIN28B (BKD), or scramble control shRNA (SC). The blastocysts treated with lentiviral particles were surgically transferred to synchronized ewes at day 9 of estrus. The conceptuses were collected at day 16 of gestation and trophectoderm (TE) was separated from embryo. LIN28A and LIN28B mRNAs and proteins were quantified by real-time PCR and Western blot. LIN28A mRNA and protein was significantly reduced in AKD TE while LIN28B mRNA and protein was significantly reduced in BKD TE compared to SC (Figure 1A,B). As expected, due to reduced LIN28, *let-7* miRNAs (*let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, let-7i*) were significantly higher in AKD and BKD TE compared to SC, and there was no significant change in *let-7* miRNAs between AKD and BKD TE (Figure 1C). These results suggest that reduced LIN28A or LIN28B led to significant increase in *let-7* miRNAs.

**Figure 1.** LIN28A or LIN28B knockdown and *let-7* miRNAs in day 16 sheep TE. (**A**) *LIN28A* and *LIN28B* mRNA in AKD (*n* = 5) and BKD (*n* = 6) day 16 TE compared to SC (*n* = 6). (**B**) Representative immunoblots for LIN28A, LIN28B, and β-actin in AKD, BKD, and SC day 16 TE, and densitometric analysis. (**C**) *Let-7* miRNAs in AKD and BKD day 16 TE and SC. \* *p* < 0.05 vs. SC.

To determine the effect of LIN28 knockdown on conceptus elongation, we measured the length of TE. Knockdown of LIN28A or LIN28B resulted in a significant reduction in day 16 TE length compared to SC (Figure 2A,B). There was no significant difference in elongation of AKD vs. BKD TE. This data suggest knockdown of either LIN28A or LIN28B in vivo resulted in reduced proliferation of trophoblast cells.

**Figure 2.** (**A**) Conceptus length at day 16 after LIN28A knockdown (AKD, *n* = 6) and LIN28B knockdown (BKD, *n* = 8) compared to scramble control (SC, *n* = 5). (**B**) Representative images of day 16 sheep conceptuses for AKD, BKD, and SC day 16 TE; \* *p* < 0.05 vs. SC.

Due to the potential for reduced proliferation of trophoblast cells in the AKD and BKD conceptuses, we measured the mRNA and protein levels of *let-7*-regulated proliferation-associated genes. The mRNA and protein levels of IGF2BP1, IGF2BP2, IGF2BP3, HMGA1, ARID3B, and c-MYC were significantly reduced in AKD and BKD day 16 TE compared to SC (Figure 3, Figure 4). These results suggest that high *let-7* miRNAs in AKD and BKD TE led to a significant reduction in expression of IGF2BP1, IGF2BP2, IGF2BP3, HMGA1, ARID3B, and c-MYC, and the reduced proliferation of trophoblast cells in AKD and BKD TE is due to significantly reduced expression of these proliferation-associated genes.

**Figure 3.** *IGF2BP1, IGF2BP2, IGF2BP3, HMGA1, ARID3B,* and *c-MYC* mRNA in AKD and BKD day 16 TE compared to SC (*n* = 5), \* *p* < 0.05 vs. SC.

**Figure 4.** Representative immunoblots for IGF2BP1, IGF2BP2, IGF2BP3, HMGA1, ARID3B, c-MYC, and β-actin, and densitometric analysis of immunoblotting results in AKD and BKD day 16 sheep TE compared to SC (*n* = 3), \* *p* < 0.05 vs. SC.
