Combination of the somatic cell nuclear transfer method and RNAi technology for the production of a prion gene-knockdown calf using plasmid vectors harboring the U6 or tRNA promoter

Abstract

By combining RNAi technology with SCNT method, we attempted to produce transgenic calves with knocked down bPRNP for technological assessments. The respective utilities of type II (tRNA) and type III (hU6) Pol III promoters in mediating plasmid vector-based RNAi for the production of a bPRNP-knockdown calf were compared. Plasmid harboring DNA for siRNA expression was introduced stably into the genome of primary cultured bovine cells. By inserting the transgenic cell into an enucleated bovine egg, SCNT embryos were produced. The ability for SCNT embryos to develop to blastocysts was higher in hU6 based vector groups (44-53%) than in a tRNA group (32%). In all, 30 hU6-embryos and 12 tRNA-embryos were transferred to 11 recipients. Only tRNA-embryos were able to impregnate recipients (6 out of 11 transfers), resulting in four aborted fetuses, one stillbirth, and one live-born calf. The expression of EGFP, a marker, was detected in all six. The bPRNP transcript levels in the nervous tissues (brain, cerebellum, spinal bulb, and spinal cord) from the calf, which was killed 20 days after birth, were reduced to 35% of those of the control calf on average, as determined by qRT-PCR. The PrPC levels, as estimated by western blot were reduced to 86% on average in the nervous tissues. These findings suggest that SCNT technology remains immature, that the tRNA promoter is useful, and that RNAi can significantly reduce PRNP mRNA levels, but insufficient reduction of PrPC levels exists in cattle under these conditions.

Figure 1

Expression of EGFP in fetal lung cells transfected with siRNA expression plasmid vector DNA and in SCNT blastocysts. Lung cells (A and B), 8-cell stage (C and D) and blastocysts (E and F). Images of A, C and E were taken under visible light. Those of B, D and F were taken under fluorescent light. Bars represent 100 µm.

Figure 2

SCNT embryo-derived transgenic calf postpartum.

Figure 3

Expression of EGFP in transgenic fetuses and calves. A–G are phase contrast images taken under automatic exposure. H–N are fluorescent images of the same fields taken with a fixed exposure time of 1.8 s. A and H are the control calf (brain); aborted fetus No. 1 (muscle) (B and I); aborted fetus No. 2 (muscle) (C and J); aborted fetus No. 3 (muscle) (D and K); aborted fetus No. 4 (muscle) (E and L); the stillborn calf (muscle) (F and M) and live-born calf (brain) (G and N). All samples were stored under −80°C. A piece of a tissue was squashed between a slide glass and a glass slip and examined. The bar in (H) shows 100 µm.

Figure 4

Relative bPRNP mRNA levels (PRNP/GAPD) in several organs of the transgenic calf as revealed by qRT-PCR. Gray columns show the control levels expressed as unity. Blank columns show those of the transgenic calf. Bars show the standard deviation of the mean. Asterisks denote a statistically significant difference from the control at less than a 5% (*) or 1% (**) level.

Figure 5

Relative PrP^C levels (PrP^C/actin) in several organs of the transgenic calf as revealed by western blotting. Gray columns show control levels expressed as unity. Blank columns show those of the transgenic calf. Bars show the range of three data obtained by application of 2.5, 5.0 and 10.0 µg proteins/lane. No statistically significant difference was found in any pair.

Figure 6

Relative PrP^C levels (PrP^C/actin) in the brain of reference cows as revealed by western blotting. The gray column shows the level of the control calf and blank columns, levels of reference cows. Bars show the average of two data obtained by application of 5.0 and 10.0 µg proteins/lane. No statistically significant difference was found between the control calf and reference cows.