ФC31 Integrase-Mediated Isolation and Characterization of Novel Safe Harbors for Transgene Expression in the Pig Genome

Abstract

Programmable nucleases have allowed the rapid development of gene editing and transgenics, but the technology still suffers from the lack of predefined genetic loci for reliable transgene expression and maintenance. To address this issue, we used ФC31 integrase to navigate the porcine genome and identify the pseudo attP sites suitable as safe harbors for sustained transgene expression. The combined ФC31 integrase mRNA and an enhanced green fluorescence protein (EGFP) reporter donor were microinjected into one-cell zygotes for transgene integration. Among the resulting seven EGFP-positive piglets, two had transgene integrations at pseudo attP sites, located in an intergenic region of chromosome 1 (chr1-attP) and the 6th intron of the TRABD2A gene on chromosome 3 (chr3-attP), respectively. The integration structure was determined by TAIL-PCR and Southern blotting. Primary fibroblast cells were isolated from the two piglets and examined using fluorescence-activated cell sorting (FACS) and enzyme-linked immunosorbent assay (ELISA), which demonstrated that the chr1-attP site was more potent than chr3-attP site in supporting the EGFP expression. Both piglets had green feet under the emission of UV light, and pelleted primary fibroblast cells were green-colored under natural light, corroborating that the two pseudo attP sites are beneficial to transgene expression. The discovery of these two novel safe harbors for robust and durable transgene expression will greatly facilitate the use of transgenic pigs for basic, biomedical and agricultural studies and applications.

Keywords: pig; pseudo attP site; safe harbor; transgene; ФC31 integrase.

Figure 1

Creation of transgenic pigs mediated by ФC31 integrase-catalyzed site-specific recombination. (a) The catalyzing principle of intra-molecular recombination between cis-positioned attP and attB sites mediated by ФC31 integrase. The pBCPB⁺ plasmid would give rise to two independent smaller plasmids due to the recombination, resulting in two novel hybrid sites, attR and attL; (b) Recombination efficiency of ФC31 integrase in the porcine embryos. End-point PCR was performed to examine the production of hybrid sites and quantified to calculate the efficiency. The TFRC gene was used as an internal control. Gradient microinjection dosages are shown; (c) The catalyzing principle of intra-molecular recombination between trans-positioned attP and attB sites mediated by ФC31 integrase. The sequence (GFP cassette) between trans-positioned attP and attB sites will be excised and the newly joined RFP cassette will be activated; (d) Activation of RFP in the porcine embryos by the co-injection of the ФC31 integrase mRNA and the reporter plasmid. Left, bright field. Middle, green fluorescence field. Right, red fluorescence field; (e) The recombination principle of attB and pseudo attP sites in mammalian cells mediated by ФC31 integrase. A plasmid containing attB will integrate into the host genome at the pseudo attP site, creating the attR’ and attL’ sites, which forms the unidirectional and single-copy transgenic structure; (f) Isolation of the integration sites in the newborn piglets using TAIL-PCR. Two pseudo attP sites were identified and mapped to the given locus in the pig genome; a star denotes the TAIL-PCR product; red arrow denotes the attB sequence and green arrow denotes the pseudo attP sequence; red bar denotes the location of pseudo attP site; colored letters denote different nucleotide bases; (g) Southern blotting was conducted to validate the recombination at the two pseudo attP sites. Specific bands are indicated by arrows; (h) The image of the transgenic piglets. Piglet numbers ^#26 and ^#61 were the two produced by the ФC31 integrase-mediated site-specific integration.

Figure 2

Characterization of porcine pseudo attP sites as safe harbors. (a) Image of the feet of EGFP-positive piglets. Red arrows indicate green fluorescence; next to ^#61 is a control foot from a wild-type pig; (b) Isolation of primary fibroblast cells from the ear tissues and sorted GFP cells. From top to bottom: the images of hair follicles under bright field, hair follicles under fluorescence field, and sorted GFP cells under fluorescence field; scale bar, 10 μm; (c) FACS analysis showing the percentage of GFP-positive cells in the population of the primary cells isolated from EGFP-positive piglets; (d) The intracellular GFP concentration detected by ELISA; (e) Green appearance of sorted GFP-positive cells under natural light when collected in DPBS solution. WT, wild-type.