1. Biochemistry and Chemical Biology
  2. Microbiology and Infectious Disease
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Novel transgenic pigs with enhanced growth and reduced environmental impact

  1. Xianwei Zhang
  2. Zicong Li
  3. Huaqiang Yang
  4. Dewu Liu
  5. Gengyuan Cai
  6. Guoling Li
  7. Jianxin Mo
  8. Dehua Wang
  9. Cuili Zhong
  10. Haoqiang Wang
  11. Yue Sun
  12. Junsong Shi
  13. Enqin Zheng
  14. Fanming Meng
  15. Mao Zhang
  16. Xiaoyan He
  17. Rong Zhou
  18. Jian Zhang
  19. Miaorong Huang
  20. Ran Zhang
  21. Ning Li
  22. Mingzhe Fan
  23. Jinzeng Yang
  24. Zhenfang Wu  Is a corresponding author
  1. South China Agricultural University, China
  2. Guangdong Wens Foodstuff Group Co., Ltd, China
  3. China Agricultural University, China
  4. University of Guelph, Canada
  5. University of Hawaii at Manoa, United States
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Cite as: eLife 2018;7:e34286 doi: 10.7554/eLife.34286
4 figures, 2 tables and 12 additional files

Figures

Figure 1 with 5 supplements
Presence of the transgene in TG pigs.

(A) DNA construct that was integrated into the pig genome for expression of the transgenic fusion enzyme in saliva. mPSP: Mouse parotid secretory protein promoter. BGH: Bovine growth hormone polyadenylation signal. Total length: 19,886 bp. (B) Expression of EGFP in the whole body of TG pigs. (C) Expression of EGFP in the heart, tongue, kidney, muscle, submandibular gland, spleen, lung, and liver of TG pigs. (D) Southern blot analysis of multi-enzyme transgene integration in TG pigs. 0.5 c, 1 c, 2 c, 3 c, and 5 c represent copy number of transgenic vector used as loading controls. The probe is shown in Figure 1A. Blank: Blank control (ddH2O).

https://doi.org/10.7554/eLife.34286.003
Figure 1—figure supplement 1
Characterization of three xylanases (XYNB, XYL11, and XYN63) expressed in the PK15 cells.

(A) Determination of the optimal pH for the activity of three xylanases. (B) The effect of pH on the stability of three xylanases. (C–E) The effects of pepsin and trypsin treatment on the stability of three xylanases. Data are expressed as the mean ±S.D. (n = 6).

https://doi.org/10.7554/eLife.34286.004
Figure 1—figure supplement 2
Characterization of two phytases (CAPPA and EAPPA) expressed in the PK15 cells.

(A) The optimal pH of CAPPA. (B) The optimal pH of EAPPA. (C) The effect of pepsin treatment on the stability of these two phytases. (D) The effect of trypsin treatment (with or without EDTA) on the stability of two phytases. Data are expressed as the mean ± S.D. (n = 3).

https://doi.org/10.7554/eLife.34286.005
Figure 1—figure supplement 3
Construction of a BgEgXyAp polycistron and its enzymatic activity in PK15 cells.

(A) Vector construct for the expression of the BgEgXyAp fusion enzyme in PK15 cells. CMV: The promoters of cytomegaloviruses. BGH: Bovine growth hormone polyadenylation signal. (B) The relative expression level of each of the fusion enzymes compared to their respective monomeric enzyme in PK15 cells. The expression level of each monomeric enzyme is normalized to that of the corresponding fusion enzyme. (C) Difference in the activity of β-glucanase, xylanase, and phytase activity detected at the indicated pH between the fusion enzyme and monomeric enzyme in PK15 cells. The activity of each monomeric was normalized to that of the corresponding fusion enzyme. Mo: Monomeric enzyme. Re: Each recombinant enzyme of the fusion enzyme. Data are expressed as the mean ± S.D. (n = 3).

https://doi.org/10.7554/eLife.34286.006
Figure 1—figure supplement 4
Genotyping analysis of founder TG pigs.

(A) and (B) PCR analysis of BgEgXyAp transgenic pig founders. AmpR: Ampicillin resistance gene in the TG vector. hyPBase: PiggyBac transposase gene. mPSP: Mouse parotid secretory protein promoter. B: Blank control (ddH2O). (C) Southern blotting analysis of the first batch of BgEgXyAp transgenic pig founders. M: Marker. 6 c, 10 c, and 4 c represent copy number of the transgenic vector that was used as loading control. N: negative control (genomic DNA of wild-type pig).

https://doi.org/10.7554/eLife.34286.007
Figure 1—figure supplement 5
Determination of the linked transgene (BgEgXyAp) copy number in the transgenic (TG) founders vs wild (WT) pigs.

Blank: ddH2O. Primers for absolute quantitative real-time PCR: Eg1314-F/R, Data are expressed as the mean ± S.D. (3 ≤ n ≤ 9).

https://doi.org/10.7554/eLife.34286.008
Figure 2 with 2 supplements
Expression and enzymatic activity of transgenes in TG pigs.

(A) RT-PCR assay for mRNA expression profiles of transgenes in different tissues. Forward primers and reverse primer are bound to the bg17A gene and eappA gene, respectively (arrows are shown in Figure 1A). Mock: Blank control (ddH2O). (B) Western blotting assay demonstrating the expression of BG17A, XYNB, and EAPPA in the saliva of TG pigs. Saliva samples were either incubated with PNGase F (+) or mock (-)-treated prior to western blotting to analyze the glycosylation status of the transgenic enzymes. PNGase F: Peptide N-glycosidase F. (C–E) Salivary β-glucanase, xylanase, and phytase activity assays of the TG pigs. (F) Concentration of total salivary protein of the TG and WT pigs. C1, C2: Age- and body weight-matched WT pigs. The data presented in the figure (C-E) can be found in Figure 2—source datas 14.

https://doi.org/10.7554/eLife.34286.009
Figure 2—source data 1

Salivary β-glucanase activity assays of TG pigs.

https://doi.org/10.7554/eLife.34286.012
Figure 2—source data 2

Salivary xylanase activity assays of TG pigs.

https://doi.org/10.7554/eLife.34286.013
Figure 2—source data 3

Salivary phytase activity assays of TG pigs.

https://doi.org/10.7554/eLife.34286.014
Figure 2—source data 4

Concentration of total salivary protein of TG and WT pigs.

https://doi.org/10.7554/eLife.34286.015
Figure 2—figure supplement 1
Assay for mRNA expression levels of the linked transgenes (BgEgXyAp) in different organs and tissues of transgenic founder pigs.

(A) RT-PCR assay for mRNA expression profiles of the transgene in different tissues. (B) Quantitative RT-PCR assay for mRNA expression levels of the linked transgenes (BgEgXyAp) in different organs and tissues of the transgenic founder pigs. Primers for relative quantitative real-time PCR: Eg1314-F/R; data are expressed as the mean ± S.D. (n = 6).

https://doi.org/10.7554/eLife.34286.010
Figure 2—figure supplement 2
Characterization of transgenic (TG) (Line2) enzymes secreted in saliva.

(A) Pattern of salivary secretion from parotid gland at different time points. A–d, Data are expressed as the mean ± S.D. (4 ≤ n ≤ 8). Values at the top of the column with different superscript letters indicate statistically significant differences (one-way ANOVA, p<0.05). (B) TG enzyme production in oral and parotid saliva at different time points. Bf, before feeding; Af, after feeding; Rt, rest time; Ft: feeding time. Data are expressed as the mean ± S.D. (n = 6).

https://doi.org/10.7554/eLife.34286.011
Comparison of the apparent total tract nutrient digestibility values (%) and fecal nutrient output (% of their dietary intake) between transgenic (TG) grower pigs (Line2) and their wild-type (WT) littermates fed on corn and soybean meal (CS) and wheat- and corn and soybean meal (WCSB)-based diets with and without exogenous feed enzymes.

(A) Comparison of the apparent total tract nutrient digestibility values (%) of dry matter (DM), crude protein (CP), phosphorus (P), and calcium (ca). (B) Comparison of fecal N, P, and Ca output. WT(+): WT grower pigs fed on the CS and WCSB diets supplemented with an optimal dose of β-glucanase, xylanase, and phytase. Data are expressed as the least square means (Lsmean ± SEM). a,b,c Values on the bar graph with different superscript letters differ significantly (ANCOVA, p<0.05). The source data are presented in Figure 3—source datas 16.

https://doi.org/10.7554/eLife.34286.020
Figure 3—source data 1

Comparison of the apparent total tract nutrient digestibility values (%) of dry matter(DM), crude protein(CP), Phosphorus(P) and calcium(ca).

https://doi.org/10.7554/eLife.34286.021
Figure 3—source data 2

Comparison of the apparent total tract nutrient digestibility values (%) between transgenic (TG) grower pigs and their wild-type (WT) littermates fed corn and soybean meal (CS diet).

https://doi.org/10.7554/eLife.34286.022
Figure 3—source data 3

Comparison of the apparent total tract nutrient digestibility values (%) between transgenic (TG) grower pigs and their wild-type (WT) littermates fed wheat, corn and soybean meal (WCSB diet).

https://doi.org/10.7554/eLife.34286.023
Figure 3—source data 4

Comparison of fecal N, fecal P and fecal Ca output.

https://doi.org/10.7554/eLife.34286.024
Figure 3—source data 5

Source Data of fecal N, fecal P and fecal Ca output by transgenic (TG) grower pigs and their wild-type (WT) littermates fed corn and soybean meal (CS)based diets with and without exogenous feed enzymes.

https://doi.org/10.7554/eLife.34286.025
Figure 3—source data 6

Source Data of fecal N, fecal P and fecal Ca output by transgenic (TG) grower pigs and their wild-type (WT) littermates fed wheat, corn and soybean meal (WCSB) based diets with and without exogenous feed enzymes.

https://doi.org/10.7554/eLife.34286.026
Growth performance of F2 TG pigs (line2) and WT littermates during the growing period from 30 kg to 115 kg.

(A) Comparison of average daily feed intake (ADFI). (B) Comparison of average daily gain (ADG). (C) Comparison of feed/gain (F/G). (D) Comparison of days to market. Data are expressed as the least square means (Lsmean ± SEM), asterisks indicate significant differences between TG and WT pigs within one line (ANCOVA, **p<0.01). The source data are presented in Figure 4—source datas 13.

https://doi.org/10.7554/eLife.34286.029
Figure 4—source data 1

Growth performance of F2 TG pigs (boars + gilts)and WT littermates during the growing period from 30 kg to 115 kg.

https://doi.org/10.7554/eLife.34286.030
Figure 4—source data 2

Growth performance of F2 TG and WT boars during the growing period from 30 kg to 115 kg.

https://doi.org/10.7554/eLife.34286.031
Figure 4—source data 3

Growth performance of F2 TG and WT gilts during the growing period from 30 kg to 115 kg.

https://doi.org/10.7554/eLife.34286.032

Tables

Table 1
Salivary secretion and the transgene enzyme activities from the unilateral parotid gland of transgenic (TG) pigs (Line2) during the grower (92–96 days old; estimated body weight: 42–45 kg) and finisher (159–191 days old; estimated body weight: 100–115 kg) phases of growth.
https://doi.org/10.7554/eLife.34286.016
ItemGrowth stagesPooled
SEM
P values
GrowerFinisher
Saliva secreted *
Saliva secretion rate (mL/min·pig)
8.4816.97**0.25<0.0001
Saliva secretion volume
(mL/kg diet consumed)
407.66**151.705.33<0.0001
Enzymes activity secreted
(U/mL saliva)
β-glucanase5.726.07**0.05<0.0001
Xylanase5.926.19**0.050.0005
Phytase7.26.870.140.1033
Enzyme activity secreted
(U/kg diet consumed)
β-glucanase2,331.84**920.8230.7<0.0001
Xylanase2,413.38**939.0331.73<0.0001
Phytase2,935.19**1,042.1938.29<0.0001
  1. *Saliva samples were collected daily from two transgenic growing pigs and two transgenic finishing pigs at 9:00 and 16:00, respectively, over the four-day period. Values are expressed as the mean and pooled SEM (n = 16 repeated sampling and measurements).

    †Means and pooled SEM (n = 16) repeated sampling and measurements). Asterisks indicate significant differences between the grower and the finisher phases within the same row (Unpaired t-test, **p<0.01).

Table 1–Source data 1

Salivary secretion from the unilateral parotid gland in the growing transgenic (TG) pigs and wild type (WT) pigs during the grower phase (92–96 days old; estimated body weight of 42–45 kg).

https://doi.org/10.7554/eLife.34286.017
Table 1–Source data 2

Salivary secretion from the unilateral parotid gland in the transgenic (TG) pigs during the finisher phase (159–191 days old; estimated body weight of 100–115 kg).

https://doi.org/10.7554/eLife.34286.018
Table 1–Source data 3

The transgene enzyme activities from the unilateral parotid gland in the transgenic (TG) pigs during the grower (92–96 days old; estimated body weight of 42–45 kg) and the finisher phases (159–191 days old; estimated body weight of 100–115 kg).

https://doi.org/10.7554/eLife.34286.019
Table 2
Comparison of the serum biochemical endpoints in the F1 transgenic (TG) grower gilts (Line2) and the wild-type (WT) gilts (50 kg) fed on the LNHP diet in Supplementary file 5.
https://doi.org/10.7554/eLife.34286.027
Serum componentTG
(n = 10)
WT
(n = 22)
Pooled SEMP value
Alkaline phosphatase (U/L)84.90**155.7212.38<0.01
Total phosphorus (Pi) (mmol/L)3.05**2.090.13<0.01
Total calcium (Ca) (mmol/L)2.532.485.510.52
Urea N (mmol/L)3.273.780.390.39
Glucose (mmol/L)4.97**3.780.21<0.01
Uric acid (µmol/L)6.307.450.540.13
D-Xylose (mmol/L)0.390.350.010.07
Total protein (g/L)59.8756.552.160.35
Zn (µmol/L)4.125.230.820.42
  1. Samples were collected at the end of the experiment.Asterisks (**) indicate significant differences at p<0.01 (unpaired t-test) between TG and WT pigs within the same row.

Table 2–Source data 1

Comparison of the serum biochemical composition in the F1 transgenic (TG) grower gilts and the wild-type (WT) gilts (50 kg) fed the low non-starch polysaccharide (NSP) diet.

https://doi.org/10.7554/eLife.34286.028

Additional files

Supplementary file 1

Integration site analysis of the TG pigs

https://doi.org/10.7554/eLife.34286.033
Supplementary file 2

Ingredients and nutrient composition of corn-cottonseed meal-rapeseed meal-soybean meal-based (CS) and wheat-corn-soybean meal-based (WCSB) diets for examining the efficiency of nutrient utilization in transgenic grower pigs (weight range: 35–54 kg)

https://doi.org/10.7554/eLife.34286.034
Supplementary file 3

Comparison of the apparent total tract nutrient digestibility values (%) between transgenic (TG) grower pigs (Line2) and their wild-type (WT) littermates fed on corn and soybean meal (CS)- and wheat, corn, and soybean meal (WCSB)- based diets with and without exogenous feed enzymes.

https://doi.org/10.7554/eLife.34286.035
Supplementary file 4

Comparison of efficiency of dietary nitrogen (N), phosphorus (P), and calcium (Ca) retention (% of their dietary intake) between transgenic (TG) grower pigs (Line2) and their wild-type (WT) littermates fed on the CS and corn-soybean (CS) or wheat-corn-soybean-bran (WCSB) diets with and without exogenous feed enzymes.

https://doi.org/10.7554/eLife.34286.036
Supplementary file 5

Ingredients and nutrient composition of the low nitrogen level and high proportion of phytate (78.4%) (LNHP) diet used to assess growth performances of the F1 transgenic (TG) grower gilts and the wild-type (WT) grower gilts (weight range: 30–50 kg).

https://doi.org/10.7554/eLife.34286.037
Supplementary file 6

Comparison of the growth performances of the F1 transgenic (TG) gilts (Line2) and the wild-type (WT) gilts fed on a low non-starch polysaccharide (NSP) diet during the growing period (weight range: 30–50 kg)

https://doi.org/10.7554/eLife.34286.038
Supplementary file 7

Ingredients and nutrient composition of the experimental diets that were formulated to determine the growth performance of the F2 transgenic (TG) grower-finisher pigs

https://doi.org/10.7554/eLife.34286.039
Supplementary file 8

Primers used in PCR and probes in southern blotting

https://doi.org/10.7554/eLife.34286.040
Supplementary file 9

Primers used in reverse transcription PCR, quantitative real-time PCR, and absolute quantitative real-time PCR

https://doi.org/10.7554/eLife.34286.041
Supplementary file 10

Antibody used in western blotting

https://doi.org/10.7554/eLife.34286.042
Supplementary file 11

Efficiency of piggyBac-mediated transgenisis to produce single-copy quad-cistronic transgenic pigs

https://doi.org/10.7554/eLife.34286.043
Transparent reporting form
https://doi.org/10.7554/eLife.34286.044

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