A pipeline for the generation of shRNA transgenic mice

Abstract

RNA interference (RNAi) is an extremely effective tool for studying gene function in almost all metazoan and eukaryotic model systems. RNAi in mice, through the expression of short hairpin RNAs (shRNAs), offers something not easily achieved with traditional genetic approaches-inducible and reversible gene silencing. However, technical variability associated with the production of shRNA transgenic strains has so far limited their widespread use. Here we describe a pipeline for the generation of miR30-based shRNA transgenic mice that enables efficient and consistent targeting of doxycycline-regulated, fluorescence-linked shRNAs to the Col1a1 locus. Notably, the protocol details crucial steps in the design and testing of miR30-based shRNAs to maximize the potential for developing effective transgenic strains. In all, this 14-week procedure provides a fast and cost-effective way for any laboratory to investigate gene function in vivo in the mouse.

Figure 1

Overview of transgenic shRNA mouse production. The figure shows the process to design, clone and test shRNAmirs for transgenic ES cells and mouse production. The expected time for completion of each stage is indicated on the right.

Figure 2

shRNAmir design and cloning. (a) Example of shRNA guide strand predictions (from DSIR or similar tool) screened against a series of Sensor exclusion criteria (gray box). The example shows two potential 21-mer predictions targeting the Renilla luciferase cDNA. Each shRNAis given a numerical designation (e.g., Ren.713) that reflects the first nucleotide position of the target sequence in the mRNA transcript. Sequences that pass all criteria are selected for cloning and testing in vitro. (b) Schematic overview of the process to transform 21-mer guide strand predictions into miR30-based cloning templates for PCR (upper box) or linker (lower box) cloning. First, the 21-mer guide strand (gray) is reverse complemented to generate the 21-mer sense strand (or 21-mer target site; green). To generate the appropriate shRNAmir template, the nucleotide immediately 5′ to the 21-mer sense strand (orange) is changed according to the nucleotide 5′ to the 21-mer target site in the mRNA transcript (gray); if the 5′ nucleotide in the mRNAis an A or U, the first base of the 22-mer sense strand becomes a C, and if the 5′ nucleotide in the mRNAis a C or G, the first base of the 22-mer sense strand becomes an A. The final 22-mer sense strand is then inserted into a 97-mer (PCR) or 110-mer (linker) cloning template (Table 1). The 22-bp guide strand is the exact reverse complement of the 22-bp target site. XhoI/EcoRI cloning fragments are then generated by PCR amplification using specific primers (Table 1) or by annealing two complementary oligonucleotides (linker cloning). Pos., position.

Figure 3

Retroviral and targeting constructs. Schematic representation of miR30-based retroviral (LMP, TGMP) and Col1a1-targeting (cTGM, cTtGM and cTtRM) vectors used in this protocol. Constructs are shown as they appear after genomic integration. TGMP is cloned within a self-inactivating (SIN) retroviral backbone and when copied into the genome the 5′ LTR promoter activity is disrupted—represented by blunted arrows. The three Col1a1-targeting constructs shown differ only in the inducible promoter (TRE or TREtight) and fluorescent spacer before shRNAmir (GFP or turboRFP).

Figure 4

shRNA genotyping and transgenic breeding. (a) Schematic representation of transgenic Col1a1-shRNAmir genotyping approach. A specific forward primer, designed to overlap the loop and guide strand of the shRNA, is used in combination with one of two common primers (RBG-R1 and RBG-R2) to generate shRNA-specific PCR product. (b) Breeding strategy to generate littermate control animals. Compound homozygous animals carrying one experimental shRNAmir (e.g., TG-APC.9365) and one control shRNAmir (e.g., TG-Ren.713), both at the Col1a1 locus, are crossed to mice carrying a tet-transactivator (tTA/rtTA) to generate F₁ animals that carry either the experimental or control shRNAmirs.

Figure 5

Picking ES cell clones and induction of GFP in transgenic cells. Phase-contrast images showing an example of a good ES cell clone (a) and two examples of bad or differentiated ES cell clones that should not be picked for further analysis (b). The left panel of b shows an example of a morphologically good clone (lower left) adjacent to a differentiated clone (upper right). If possible, we suggest avoiding this good clone to minimize the chance of isolating a mixed clone. (c) Phase-contrast and fluorescence images of Col1a1-targeted transgenic KH2 ES cells carrying a control (TG-Ren.713) shRNAmir. Two days after dox treatment (1 µg ml⁻¹), ES cells show strong GFP expression, as measured by microscopy. (d) Flow cytometry analysis of targeted ES cells 4 d after dox treatment. (e) Flow cytometry analysis of single-cell populations derived from intestine (left), whole bone marrow (middle) and whole spleen (right) cells. Samples represent nontransgenic (gray line) and double-transgenic CAGs-rtTA3/TG-Ren.713 mice either untreated (black fill) or dox treated (green fill). Intestine and bone marrow show quite uniform GFP expression, whereas spleen shows heterogeneous expression of the GFP-shRNAmir cassette.