Heritable and stable gene knockdown in rats

Abstract

The rat has served as an excellent model for studies on animal physiology and as a model for human diseases such as diabetes and alcoholism; however, genetic studies have been limited because of the inability to knock out genes. Our goal was to produce heritable deficiencies in specific gene function in the rat using RNA interference to knock down gene expression in vivo. Lentiviral-mediated transgenesis was used to produce rats expressing a short hairpin RNA targeting Dazl, a gene expressed in germ cells and required for fertility in mice. Germ-line transmission of the transgene occurred, and its expression correlated with significant reductions in DAZL protein levels and male sterility, and the knockdown was stable over multiple generations (F(1)-F(3)). This study demonstrates an efficient system by which directed reverse genetic analysis can now be performed in the rat.

Fig. 1.

Dazl shRNA construct design. DNA oligonucleotides containing Dazl shRNA 1 (shown) and 2 (not shown) were inserted downstream of the U6 promoter in pLLU2G. The green sequence is equivalent to the Dazl mRNA target sequence. The presumed U6 transcription start site is designated as +1, and the predicted shRNA structure is shown. The red sequence corresponds to the antisense RNA probe generated for RNase protection assay. SIN-LTR, self-inactivating long terminal repeat; Psi, HIV packaging signal; cPPT, central polypurine track; U6, Pol III promoter; shRNA, small hairpin RNA; Ubc, Ubc promoter; eGFP, enhanced green fluorescent protein; WRE, woodchuck hepatitis virus response element.

Fig. 2.

Transgene expression and DAZL knockdown. (A) Western blot analysis to detect DAZL-MYC production in cells transduced with virus carrying pLLU2G (Vector) or pLLU2G-Dazl1 (Dazl-shRNA) and transiently transfected with pCDNA-Dazl-myc. The blot was stripped and reprobed with anti-tubulin to verify equal loading of protein. (B–E) Fluorescence of wild-type (B and D) and transgenic (C and E) neonatal pups (B and C) or adult ear punch skin (D and E). (F) RNase protection assay using a probe to detect Dazl short interfering RNA (from Dazl shRNA 1) in testis RNA from transgenic rats (17-9 or 16-13 Tg), a wild-type sibling of the 17-9 transgenic rat (Wt), or yeast RNA (mock). The protected portion of the antisense probe sequence is shown in red in Fig. 1. The sense probe is reverse-complementary to the antisense probe. Addition of RNase is indicated by +. (G) Western blot analysis to detect Dazl, Tex11, tubulin, and GFP expression in the testis. Lanes were loaded as follows. (Left) First lane, transgenic 16-13; second lane, wild-type sibling of 17-9 F₁; third and fourth lanes, two transgenic 17-9 F₁s. (Right) First lane, wild-type sibling of 17-9 F₂; second lane, transgenic 17-9 F₂. (H–K) Immunofluorescence to detect DAZL protein in testis cryosections of a Dazl-shRNA rat (H) and a wild-type sibling (J). DAPI (DNA) staining of the same sections is also shown (I and K).

Fig. 3.

Analysis of Dazl-shRNA phenotype. (A–C, E–G, and I) Hematoxylin/eosin histological staining of testis (A, B, E, and F) and epididymis (C and G) from an adult (9-week-old) Dazl-shRNA rat (E–G) and a wild-type sibling (A–C). (D and H) CREM immunofluorescence (red) and overlaid DAPI staining (blue) in testis of a young (6-week-old) Dazl-shRNA rat (H) or a wild-type sibling (D). [Scale bars: 50 μm in A (for A, C–E, and G–I) and 10 μm in B (for B and F).] (I) Hematoxylin/eosin histological staining of testis from a 6-month-old Dazl-shRNA rat. (J) Testis and epididymis from 6-month-old wild-type (Upper) or Dazl-shRNA (Lower) rat. (K) Graphical representation of mean testis weight (in grams) at various ages. The following numbers of testes were examined: at 6 weeks old, 4 wild-type and 10 transgenic; at 7 weeks old, 2 wild-type and 6 transgenic; at 9 weeks old, 2 wild-type and 4 transgenic; at 11 weeks old, 4 wild-type and 10 transgenic; at 26 weeks old, 2 wild-type and 4 transgenic.