Fig 1.
GM milk was digested by simulated gastric fluid in vitro.
(A) Tricine-SDS-PAGE analysis and (B) Western blot analysis of the degradation of GM milk containing HBD3 in simulated gastric fluid. The blot was probed with polyclonal antibody to HBD3. Pepsin was 35 kDa, and HBD3 standards was 5 kDa. (Notably, the standard protein marker indicated by the location in gel was bigger than its real molecular weight about 5 kDa.)
Fig 2.
Body weight and daily food consumption.
(A) Mean body weight of male mice (n = 20); (B) Mean body weight of female mice (n = 20); (C) Mean daily food consumption of male mice (n = 20); (D) Mean daily food consumption of female mice (n = 20).
Fig 3.
Gastric emptying function of mice in vivo.
Percentage of phenol red in stomach in gastric emptying assay of male (A) and female (B) mice. The result was showed as the rate of the amount of phenol red in stomach/ the total amount of phenol red in stomach and small intestine.
Fig 4.
The analysis of intestinal permeability in ileum and colon of male mice.
(A) Histopathological results of HE staining. Scale bar = 50 μm. (B) The ultrastructure were examined by transmission electron microscope. TJ: tight junctions. AJ: adherens junctions. DS: desmosomes. Scale bar = 400 nm. (C) Result of immunofluorescence of zo-1, occludin, and claudin-1 in intestinal epithelial cells. Frozen sections of ileum and colon were labeled for zo-1 (red), occludin (red), claudin-1 (red) and nuclei (blue). Scale bar = 100 μm. (D) Tracer experiment to examine the permeability in ileum and colon. The green fluorescence signals of biotin were found to be restricted to the lumen of the ileum and colon in each group. Scale bar = 50 μm.
Fig 5.
DGGE electrophoretogram of 16S rDNA PCR products from intestinal content samples.
The numbers 1 to 25 on the top of the electrophoretogram indicated as following: the 1–5 represented the samples of duodenum of 30G, 30N, 10G, 10N, and C groups. 6–10 represented the samples of jejunum of 30G, 30N, 10G, 10N, and C groups. 11–15 represented the samples of ileum of 30G, 30N, 10G, 10N, and C groups. 16–20 represented the samples of cecum of 30G, 30N, 10G, 10N, and C groups. 21–25 represented the samples of colon of 30G, 30N, 10G, 10N, and C groups. Every sample was collected from 5 mice to treat as a sample pool.
Fig 6.
The species of microflora in duodenum, jejunum, ileum, cecum and colon of 30G, 30N, 10G, 10N and C groups without regarding to gender.
(A) Phyla and (B) genus distribution of 16S rDNA clones derived from intestinal content samples. The numbers 1 to 25 were labeled as in Fig 5.
Fig 7.
Cluster analysis of 16S rDNA clones by UPMGA.
Numbers along the top indicated the similarity coefficient. The similarity coefficient of the adjacent two samples was labeled in red. The numbers 1 to 25 were labeled as those in Fig 5. A represented the cluster analysis of 16S rDNA clones of all samples, and B-F represented cluster analysis of 16S rDNA clones derived from duodenum, jejunum, ileum, cecum, and colon, respectively.
Fig 8.
Shannon diversity analysis of intestinal microflora diversity in 30G, 30N, 10G, 10N and C groups.
Fig 9.
Intestinal contents and main organs were used for HGT detection by PCR.
(A) DNA of transgenic cattle was used as template in PCR. Lane 1 represented the PCR was used HBD3 primers, and amplicon size was 1156bp. Lane 2 represented the PCR was used HBD3-CSN primers, and amplicon size was 415bp. M: 100 bp DNA marker. (B) HGT was detected by PCR with DNA of intestinal contents as template. 0–6 represented transgenic cattle, duodenum, jejunum, ileum, cecum, colon, and ddH2O, respectively. Bacterial 16S rDNA was amplified with bacteria universal primers (GC-338F and 518R), and amplicon size was 200bp. (C) HGT was detected by PCR with DNA of tissue of main organs as template. 0′-9′represented transgenic cattle, heart, liver, spleen, lung, kidney, large intestine, small intestine, muscle, and ddH2O, respectively. β-actin was amplified with β-actin primers, and amplicon size was 241bp. HBD3 was amplified with HBD3 primers. No product at 1156bp position was found in both 30G and 10G of intestinal contents and main organs. HBD3-CSN was amplified with HBD3-CSN primers. No product at 415bp position was found in both 30G and 10G of intestinal contents and main organs.