Generation of Multi-Transgenic Pigs Using PiggyBac Transposons Co-expressing Pectinase, Xylanase, Cellulase, β-1.3-1.4-Glucanase and Phytase

Abstract

The current challenges facing the pork industry are to maximize feed efficiency and minimize fecal emissions. Unlike ruminants, pigs lack several digestive enzymes such as pectinase, xylanase, cellulase, β-1.3-1.4-glucanase, and phytase which are essential to hydrolyze the cell walls of grains to release endocellular nutrients into their digestive tracts. Herein, we synthesized multiple cellulase and pectinase genes derived from lower organisms and then codon-optimized these genes to be expressed in pigs. These genes were then cloned into our previously optimized XynB (xylanase)- EsAPPA (phytase) bicistronic construct. We then successfully generated transgenic pigs that expressed the four enzymes [Pg7fn (pectinase), XynB (xylanase), EsAPPA (phytase), and TeEGI (cellulase and β-glucanase)] using somatic cell cloning. The expression of these genes was parotid gland specific. Enzymatic assays using the saliva of these founders demonstrated high levels of phytase (2.0∼3.4 U/mL) and xylanase (0.25∼0.42 U/mL) activities, but low levels of pectinase (0.06∼0.08 U/mL) activity. These multi-transgenic pigs are expected to contribute to enhance feed utilization and reduce environmental impact.

Keywords: PiggyBac; digestive enzymes; polycistronic; salivary gland; transgenic pigs.

FIGURE 1

Characterization of the three pectinase genes expressed in PK-15 cells. Pectinase activities of PgaA, Pg7fn, and PGI were evaluated using 1% (A) polygalacturonic acid, (B) 55∼70% esterified pectin, and (C) >85% esterified pectin as substrates at pH 4.5, respectively. (D) Pectinase activities of Pg7fn and PGI at different pH levels (1.0∼8.0). (E) Pg7fn and PGI were incubated with different pepsin and trypsin pH solutions at 39.5°C for 2 h. Control represents the pcDNA 3.1(+) vector. Data were shown as mean ± SEM, n = 3 (one-way ANOVA). ^∗P < 0.05, ^∗∗P < 0.01.

FIGURE 2

Characterization of six cellulase genes expressed in PK-15 cells. (A) Cellulase and (B) β-glucanase activities of cel5B, egII, AG-egaseI, TeEGI, cel9, and Bh-egaseI were evaluated at suitable pH conditions. (C) Cellulase activities of egII and TeEGI at different pH levels (2.0∼7.0). (D) β-glucanase activities of egII and TeEGI at different pH levels (2.0∼7.0). (E) Cellulase and (F) β-glucanase activities of egII and TeEGI were measured following incubation with different pepsin and trypsin pH solutions. Control represents the pcDNA3.1(+) vector. Data were shown as mean ± SEM, n = 3 (t-test). ^∗P < 0.05 or ^∗∗P < 0.01.

FIGURE 3

Enzymatic activities between the polycistronic and single gene vector constructs. The effect of 2A linker peptide on (A) pectinase, (B) phytase, (C) cellulase, and β-glucanase activity. (D) Schematic of the PXAT vector. (E) Enzymatic activities between PXAT and its corresponding protein expressed by single gene constructs. (F) Relative mRNA expression levels between genes expressed with PXAT and single gene constructs. Control represents the pcDNA3.1(+) vector. Data were shown as mean ± SEM, n = 3 (one-way ANOVA). ^∗P < 0.05 or ^∗∗P < 0.01.

FIGURE 4

Generation and identification of the transgenic pigs. (A) Schematic of the transgenic plasmid mPSP-PXAT. The mPSP-PXAT consisted of the mouse parotid secretory protein (mPSP) promoter, loxp system with the neo-EGFP marker protein, and a PiggyBac transposon. (B) EGFP was deleted using Cre recombinase prior to somatic cell nuclear transfer. (C) Genomic identification of transgenic piglets using PCR and gel electrophoresis. (D) Transgenic piglets at 2-week-old. (E) Southern blot analysis of transgene integration in transgenic piglets. Genomic DNA was digested using Kpn I and Eco47 III endonucleases. (F) Copy number determination in transgenic piglets by absolute quantification. (G) Western blotting analysis of XynB and TeEGI protein expression. Salivary amylase was used as a protein reference. (H) Salivary pectinase, (I) xylanase, (J) phytase, (K) cellulase, and (L) β-glucanase expression at 4 months. M is the DNA marker, P indicates mPSP-PXAT plasmid; N and WT represent wild-type pigs. Data were shown as mean ± SEM (one-way ANOVA). ^∗P < 0.05.