science.sciencemag.org/content/367/6481/eaaw8429/suppl/DC1

Supplementary Materials for

Maternal gut microbiota in pregnancy influences offspring metabolic

phenotype in mice

Ikuo Kimura*†, Junki Miyamoto*, Ryuji Ohue-Kitano, Keita Watanabe, Takahiro

Yamada, Masayoshi Onuki, Ryo Aoki, Yosuke Isobe, Daiji Kashihara, Daisuke Inoue,

Akihiko Inaba, Yuta Takamura, Satsuki Taira, Shunsuke Kumaki, Masaki Watanabe,

Masato Ito, Fumiyuki Nakagawa, Junichiro Irie, Hiroki Kakuta, Masakazu Shinohara,

Ken Iwatsuki, Gozoh Tsujimoto, Hiroaki Ohno, Makoto Arita, Hiroshi Itoh, Koji Hase†

*These authors contributed equally to this work.

†Corresponding author. Email: ikimura@cc.tuat.ac.jp (I.K.); hase-kj@pha.keio.ac.jp (K.H.)

Published 28 February 2020, Science 367, eaaw8429 (2020)

DOI: 10.1126/science.aaw8429

This PDF file includes:

Figs. S1 to S24

Tables S1 to S5

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Fig. S1. Male pups delivered from GF mothers exhibit obesity during growth. (A) Representative photograph and body weight at postnatal day 1 (n = 10–12 animals per group). (B) Adipocyte area and representative macroscopic appearance of white adipose tissue (WAT; n = 6 tissues per group). (C) Hepatic triglycerides (TGs) and oil red O staining (n = 7, 8 tissues per group). (D) Plasma insulin levels (left, n = 8, 6 plasma samples per group), pancreatic islet area (middle, n = 8, 6 tissues per group), and representative macroscopic appearance (right). (E) Daily food intake (n = 7, 8 animals per group). Male mice were analyzed at 16 weeks of age. Student’s t-test; **P < 0.01 and *P < 0.05. All data are presented as the means ± SEM of mice from 3 litters. SPF, conventional offspring derived from SPF ICR mothers; GF, conventional offspring derived from GF ICR mothers.

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Fig. S2. Female pups delivered from GF mothers exhibit obesity during growth. (A, B) Body weight changes during HFD trial (n = 10, 9 animals) (A) and tissue weight after 12-week HFD intervention (n = 10, 9 tissues) (B). gona, gonadal; peri, perirenal; sub, subcutaneous. (C, D) Plasma glucose, TGs, non-esterified fatty acids (NEFAs), total cholesterol (C), and insulin levels (D) (n = 8–10 plasma samples per group). (E) Body temperature (n = 8 animals per group). (F) Heart rate (right, n = 10, 9 animals per group). (G) Energy expenditure of pups of SPF or GF mothers fed HFD (n = 8 animals). (H) Glucose tolerance test (GTT, left) and insulin tolerance test (ITT, right) (n = 8 animals per group). Female mice were analyzed at 16 weeks of age (B–H).

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Student’s t-test; **P < 0.01 and *P < 0.05. All data are presented as the means ± SEM of mice from 3 litters. SPF, conventional offspring derived from SPF ICR mothers; GF, conventional offspring derived from GF ICR mothers.

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Fig. S3. Postnatal gut microbiota does not directly impact the obesity of offspring of GF mothers. (A, B) Gut microbiota compositions determined as the relative abundance of microbial taxa at the family level (A) and their beta diversity as shown via principal component analysis (PCA) (B) in the feces of offspring from SPF and GF mothers during infancy (2-week-old, n = 10 fecal samples of pups from 3 litters) and adulthood (16-week-old, n = 8 fecal samples of pups from 3 litters). SPF, conventional offspring derived from SPF ICR mothers; GF, conventional offspring derived from GF ICR mothers.

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Fig. S4. Pups delivered by caesarean section from GF mothers exhibit obesity during growth. (A, B) Body weight changes during HFD trial (n = 9 animals) (A) and tissue weight after 12-week HFD intervention (n = 9 tissues) (B). epi, epididymal; peri, perirenal; sub, subcutaneous. (C, D) Plasma glucose, TGs, NEFAs, total cholesterol (C), and insulin levels (D) (n = 9 plasma samples per group). (E) Energy expenditure of pups of SPF or GF mothers fed HFD (n = 6 animals). Male mice were analyzed at 16 weeks of age (B–E). Student’s t-test; **P < 0.01 and *P < 0.05. All data are presented as the means ± SEM of mice from 3 litters. SPF, conventional offspring derived from SPF mothers; GF, conventional offspring derived from GF mothers.

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Fig. S5. Postnatal gut microbiota in caesarean offspring of GF mothers. (A, B) Gut microbiota compositions determined as the relative abundance of microbial taxa at the family level (A) and their beta diversity as shown via PCA (B) in feces of caesarean offspring of SPF and GF mothers during infancy (2-week-old, n = 8 fecal samples of pups from 3 litters) and adulthood (16-week-old, n = 8 fecal samples of pups from 3 litters). SPF, conventional offspring derived from SPF mothers; GF, conventional offspring derived from GF mothers.

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Fig. S6. Compositions of gut microbiota in mothers between ICR and C57BL/6J genetic background. (A, B) Gut microbiota compositions determined as the relative abundances of microbial taxa at the family level (A) and their beta diversity as shown via PCA (B) in feces of E18.5 pregnant ICR and C57BL/6J wild-type mice (n = 7–8 fecal samples per group).

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Fig. S7. Male pups delivered from GF mothers in C57BL/6J genetic background exhibit obesity during growth. (A, B) Body weight changes during HFD trial (n = 10, 5 animals) (A) and tissue weight after 12-week HFD intervention (n = 10, 5 tissues) (B). epi, epididymal; peri, perirenal; sub, subcutaneous. (C, D) Plasma glucose, TGs, NEFAs, total cholesterol (C), and insulin levels (D) (n = 10, 5 plasma samples per group). (E) Body temperature (n = 10, 5 animals per group). (F) Heart rate (right, n = 10, 5 animals per group). (G) Energy expenditure of pups of SPF or GF mothers fed HFD (n = 9, 5 animals). (H) Glucose tolerance test (GTT, left) and insulin tolerance test (ITT, right) (n = 7, 5 animals per group). Male mice were analyzed at 16 weeks of age (B–H). Student’s t-test; **P < 0.01 and *P < 0.05. All data are presented as the means ± SEM

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of mice from 4 litters. SPF, conventional offspring derived from SPF mothers; GF, conventional offspring derived from GF mothers.

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Fig. S8. Female pups delivered from GF mothers in C57BL/6J genetic background exhibit obesity during growth. (A, B) Body weight changes during HFD trial (n = 12, 4 animals) (A) and tissue weight after 12-week HFD intervention (n = 12, 4 tissues) (B). gona, gonadal; peri, perirenal; sub, subcutaneous. (C, D) Plasma glucose, TGs, NEFAs, total cholesterol (C), and insulin levels (D) (n = 12, 4 plasma samples per group). (E) Body temperature (n = 11, 4 animals per group). (F) Heart rate (n = 8, 4 animals per group). (G) Energy expenditure of pups of SPF or GF mothers fed HFD (n = 8, 4 animals). (H) Glucose tolerance test (GTT, left) and insulin tolerance test (ITT, right) (n = 8, 4 animals per group). Female mice were analyzed at 16 weeks of age (B–H). Student’s

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t-test; **P < 0.01 and *P < 0.05. All data are presented as the means ± SEM of mice from 4 litters. SPF, conventional offspring derived from SPF mothers; GF, conventional offspring derived from GF mothers.

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Fig. S9. Commensal bacteria in embryos or in utero. (A) Total bacterial numbers in E18.5 whole embryos, placenta, embryonic colon, and adult cecum were determined by qPCR (whole embryo, placenta, and embryonic colon: n = 8 embryos from 8 litters and n = 8 cecum samples per group). Data are presented as the mean ± SEM. (B) Culture of amniotic fluids and cecal contents from E16.5 pregnant ICR mice in BL medium or MRS medium. Representative photographs of amniotic fluids (left, n = 5 samples) and cecal contents (right, n = 5 samples) diluted with sterile PBS at 106.

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Fig. S10. Plasma metabolome of SPF and GF mice. Heat map of plasma metabolite profiles of mothers (upper) and embryos (E18.5; lower). n = 5 plasma samples of mothers and n = 5 plasma samples of embryos from 5 litters. Among mothers, 5 of 280 (hydrophilic 88 + lipophilic 192) identified metabolites showed significant differences in abundance (q < 0.05: red open square). Among embryos, 12 of 280 identified metabolites showed significant differences in abundance (q < 0.05: red open square).

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Fig. S11. Plasma SCFA concentration. Levels of plasma SCFAs during pregnancy day 14.5, 16.5, and 18.5, as determined by GC/MS (n = 8 plasma samples per group). Data are presented as the mean ± SEM.

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Fig. S12. In situ hybridization and qRT-PCR analysis of Gpr41 and Gpr43 mRNA expression in tissues. (A) Localization of Gpr41 in mouse embryos (E13.5, E16.5, and E18.5). SCG: superior cervical ganglion, ST: sympathetic trunk. TH: anti-tyrosine hydroxylase immunostaining. (B) Localization of Gpr41 in mouse embryos (E18.5). (C) Localization of Gpr43 in mouse embryos (E13.5, E15.5, E16.5, and E18.5). (D) Localization of Gpr43 in adult ileum (in 7-week-old). (E, F, G) Expression of Gpr41 and Gpr43 in SCG (E), the colon (F), and the pancreas (G) at E16.5 and E18.5. (n = 8 tissues from 4 litters per group). H&E: hematoxylin and eosin staining (A–D). Red and black grains indicate Gpr41 or Gpr43 mRNA localization (A–D). Values are shown

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relative to 18S rRNA gene expression (E, F, G). Student’s t test; **P < 0.01. Data are presented as the means ± SEM (E, F, G).

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Fig. S13. Compositions of gut microbiota and plasma SCFAs in mothers among mouse strains. (A, B) Gut microbiota compositions determined as the relative abundance of microbial taxa at the family level (A) and their beta diversity as shown via PCA (B) in feces of E18.5

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pregnant C57BL/6J wild-type, C57BL/6J-background Gpr41-/-, and C57BL/6J-background Gpr43-/- mice (n = 8 fecal samples per group). (C) Levels of plasma SCFAs in E18.5 pregnant C57BL/6J wild-type, C57BL/6J-background Gpr41-/-, and C57BL/6J-background Gpr43-/- mice (n = 8 plasma samples per group) as determined by GC/MS. Data are presented as the means ± SEM.

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Fig. S14. Functions of embryonic SCFA receptors in SCG. (A) Effects of SCFAs (0.01, 0.1, and 1 mM) on sympathetic neuronal differentiation. TH-positive cells were counted (TH, red; nestin, green; DAPI, blue; n = 9–10 independent experiments from three biological replicates per condition). (B) Propionate-induced decrease in intracellular cAMP concentration in sympathetic neural cells from WT and Gpr41-/- mice at 10 min after 30 min of pre-treatment with 3-isobutyl-L-methylxanthine (IBMX). n = 4 independent experiments from two biological replicates per condition. (C) Propionate (1 mM)-induced sympathetic neurite outgrowth after 4 days exposure to inhibitors (NF023, 10 µM; gallein, 5 µM; or U0126, 10 μM). n = 3 independent experiments per condition. Dunnett’s test (A) and Tukey–Kramer’s (B, C); **P < 0.01 and *P < 0.05. NS, not significant. Data are presented as the mean ± SEM.

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Fig. S15. Functions of embryonic SCFA receptors. (A) Expression of colonic Pax4, Pax6, and Gcg (E18.5, upper; E16.5, lower) (n = 6–8 tissues of embryos from 3 litters per group). WT and Gpr43-/- mice (C57BL/6J). SPF and GF mice (ICR). (B) Changes in marker gene expression during enteroendocrine cell differentiation of embryonic intestinal organoids (E15.5). n = 5 independent experiments. (C) Expression of Gcg in the presence or absence of GPR43 ligands (1 and 10 mM

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propionate) after 24 h treatment of adult organoids (8 weeks old) from wild-type (left) and Gpr43-

/- mice (right). n = 5 independent experiments from two biological replicates. (D) Expression of Nkx6.1 and Ins2 in the pancreas (E18.5, left; E16.5, right). n = 6–8 tissues of embryos from 3 litters. WT and Gpr43-/- mice (C57BL/6J). SPF and GF mice (ICR). (E) Expression of Ins2, Gpr41, and Gpr43 in AR42J cells after induction of differentiation (n = 6 independent experiments from two biological replicates per condition). Values are shown relative to 18S rRNA gene expression. (F) Expression of Gpr43 in siRNA-transfected AR42J cells (n = 4 independent experiments). (G) TH expression in ICR E16.5 whole heart (n = 8 tissues of embryos from 3–4 litters per group) and representative western blots. Student’s t-test (A, B, D, G) and Tukey–Kramer’s test (C); **P < 0.01 and *P < 0.05. NS, not significant. Data are presented as the mean ± SEM.

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Fig. S16. ICR and C57BL/6J embryonic glucose homeostasis. (A) Plasma insulin (left) and plasma glucose (right) levels (n = 8 plasma samples of ICR mothers and n = 8 plasma samples of E18.5 embryos from 8 litters) (B) Plasma insulin (left) and plasma glucose (right) levels (n = 4–8 plasma samples of C57BL/6J mothers and n = 4–8 plasma samples of E18.5 embryos from 4–8 litters). Student’s t-test; **P < 0.01. Data are presented as the mean ± SEM.

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Fig. S17. Functions of embryonic SCFA receptors in Gpr41/Gpr43 double-deficient mice. (A) Schematic representation of the genomic structures of the Gpr41 and Gpr43 genes. Gpr41 and Gpr43 double-knockout (DKO) mice were generated using the CRISPR/Cas9 system in wild-type C57BL/6 zygotes. Coding and untranslated exon regions are shown as black and grey boxes and words, respectively. Guide RNA (gRNA), protospacer adjacent motif (PAM), and insert sequences are indicated by green, purple, and light blue boxes, respectively. (B) Mouse genotypes were

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determined by PCR using the indicated primers to detect wild-type and mutant alleles of Gpr41 and Gpr43. (C) Expression of cardiac TH in postnatal day 1 (P1) Gpr41-/-Gpr43-/- whole hearts (n = 11 tissues of littermates from 13 litters). (D) Expression of colonic Pax4, Pax6, and Gcg in P1 Gpr41-/-Gpr43-/- mice (n = 8–11 tissues of littermates from 11 litters). (E) Expression of pancreatic Nkx6.1 and Ins2 in P1 Gpr41-/-Gpr43-/- mice (n = 8–11 tissues of littermates from 11 litters). Student’s t-test; **P < 0.01. Data are presented as the mean ± SEM. DKO, Gpr41-/-Gpr43-/- mice.

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Fig. S18. Dietary fiber supplementation for pregnant mothers and metabolic phenotypes of offspring. (A) Body weight at postnatal day 1 (n = 9 animals from 4 litters per group). (B) Plasma insulin levels (n = 8 plasma samples from 3–4 litters per group). (C) Plasma GLP-1 levels (n = 8 plasma samples from 3–4 litters per group). (D) GTT (left) and ITT (right) (n = 8–9 animals from 4 litters per group). (E) Energy expenditure (n = 8 animals per group). Male mice were analyzed at 16 weeks of age (B–E). Student’s t-test (B, C, E) and Tukey–Kramer’s test (A, D); **P < 0.01 and *P < 0.05 (LFi vs. HFi); ##P < 0.01 and #P < 0.05 (HFi vs. HFi + Abx). Data are presented as the mean ± SEM. LFi, offspring derived from LFi ICR mothers; HFi, offspring derived from HFi ICR mothers.

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Fig. S19. Postnatal gut microbiota in offspring of high-fiber diet-fed mothers. (A, B) Gut microbiota compositions determined as the relative abundance of microbial taxa at the family level (A) and their beta diversity as shown via PCA (B) in feces of offspring of low-fiber diet or high-fiber diet fed mothers during infancy (2-week-old, n = 8 fecal samples of pups from 3 litters) and adulthood (16-week-old, n = 9 fecal samples of pups from 3 litters). LFi, offspring derived from LFi ICR mothers; HFi, offspring derived from HFi ICR mothers.

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Fig. S20. Plasma metabolome analysis during dietary fiber feeding. Heat map of plasma metabolite profiles of ICR mothers (upper) and ICR embryos (lower). n = 5 plasma samples of mother and n = 5 plasma samples of embryos from 5 litters. Among mothers, 11 of 363 (hydrophilic 153 + lipophilic 210) identified metabolites showed significant differences in abundance (q < 0.05: red open square). Among embryos, 4 of 363 identified metabolites showed significant differences in abundance (q < 0.05: red open square).

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Fig. S21. PET imaging. (A) Count per minute (cpm)/g tissue relative to that of the liver in pregnant rats by gamma counting [left, n = 6 (from 6 litters with averaged values of two kidneys and multiple embryos per litter)], and static scans 20–40 min after intestinal administration of [11C] propionate (right). Green dotted lines and arrowheads indicate embryos. Data are presented as the mean ± SEM. (B) Flowchart of [11C]-propionate synthesis. (C) Analytical HPLC charts of [11C]-propionate. Upper and lower panels show UV (210 nm) and RI time course charts of [11C]-propionate, respectively. Because the retention time at 8.046 min in the UV chart corresponds to the retention time at 8.002 min in the RI chart, the product was determined to be [11C]-propionate. Radiochemical purity and total yield were 96.5% and 5.77%, respectively. (D) Representative decay-corrected abdominal PET/CT images of pregnant rats after intestinal administration of [11C]-propionate. Static scans at (i) 0–5 min, (ii) 10–15 min, and (iii) 20–40 min after intestinal administration of [11C]-propionate. Green dotted lines indicate embryos. H, T, t, s, and c stand for head, tail, transverse, sagittal, and coronal planes, respectively. At 20 minutes after administration, the existence of the tracer was observed in the embryos indicated by arrowheads. PET/CT imaging of pregnant rats was performed 3 times. Double-ended arrow length = 5 cm.

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Fig. S22. Propionate supplementation for pregnant mothers and metabolic phenotypes of offspring. (A) Plasma propionate determined by GC/MS in the mothers and embryos (E18.5). n = 7 plasma samples of mothers and n = 7 plasma samples of embryos from 7 litters per group. (B) Plasma insulin levels (n = 8 plasma samples from 4 litters per group). (C) Plasma GLP-1 levels (n = 8 plasma samples from 4 litters per group). (D) GTT (left) and ITT (right). n = 8, 9 animals from 4 litters per group. (E) Energy expenditure (n = 8 animals from 4 litters per group). Male mice were analyzed at 16 weeks of age (B–E). Student’s t-test; ** P < 0.01 and * P < 0.05. Data are presented as the mean ± SEM. LFi, offspring derived from LFi ICR mothers; propionate, offspring derived from propionate-supplemented ICR mothers (B–E).

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Fig. S23. Postnatal gut microbiota in offspring of propionate-supplemented mothers. (A, B) Gut microbiota compositions determined as the relative abundance of microbial taxa at the family level (A) and their beta diversity as shown via PCA (B) in feces of offspring of low-fiber diet-fed or propionate-supplemented mothers during infancy (2-week-old, n = 9 fecal samples of pups from 3 litters) and adulthood (16-week-old, n = 9 fecal samples of pups from 3 litters). LFi, offspring derived from LFi mothers; propionate, offspring derived from propionate-supplemented ICR mothers.

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Fig. S24. Pups delivered from high fiber diet-fed GF mothers exhibit obesity during growth, whereas pups delivered from propionate supplemented GF mothers suppress the phenotype. (A, B) Body weight changes during HFD trial (n = 8 animals) (A) and tissue weight after 12-week HFD intervention (n = 8 tissues) (B). epi, epididymal; peri, perirenal; sub, subcutaneous. (C, D) Plasma glucose, TGs, NEFAs, total cholesterol (C), and insulin levels (D) (n = 8–9 plasma samples per group). (E) Body temperature (n = 8, 7 animals per group). (F) Heart rate (n = 8 animals per group). (G) Energy expenditure of pups of SPF or GF mothers fed HFD (n = 8 animals). (H) Glucose tolerance test (GTT, left) and insulin tolerance test (ITT, right). n = 9 animals per group. Male mice were analyzed at 16 weeks of age (B–H). Student’s t-test; **P < 0.01 and *P < 0.05.

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Data are presented as the mean ± SEM of mice from 3 litters. GF-HFi, offspring derived from HFi GF ICR mothers; GF-propionate, offspring derived from propionate-supplemented GF ICR mothers.

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Table S1. Diet composition.

Table S1.

HFD (D12492) Regular chow (CMF)

Product gm% gm%

Protein 26 27.8

Carbohydrate 26 56

Fat 35 8.3

Fiber (crude) - 3.1

Fiber (cellulose) 6 -

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Table S2. Diet composition in HFi experiments.

Table S2.

LFi

Low fiber (AIN-93G)

HFi

High fiber (10% inulin)

Product gm% gm%

Protein 20 20

Carbohydrate 63 53

Fat 7 7

Fiber (cellulose) 6 6

Fiber (inulin) 0 10

Ingredient gm gm

Casein 200 200

L-Cystine 3 3

Corn Starch 397.486 297.486

Maltodextrin 10 132 132

Sucrose 100 100

Cellulose, BW200 50 50

Orafti GR (Inulin) 0 100

Soybean Oil 70 70

t-Butylhydroquinone 0.014 0.014

Mineral Mix S10022G 35 35

Vitamin Mix V10022G 10 10

Choline Bitartrate 2.5 2.5

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Table S3. Diet composition in SCFA supplementation experiments.

Table S3.

LFi

(AIN-93G + cellulose)

Propionate

(AIN-93G + propionate)

Product gm% gm%

Protein 19 19

Carbohydrate 60 60

Fat 7 7

Fiber (cellulose) 11 6

Sodium propionate 0 5

Ingredient gm gm

Casein 200 200

L-Cystine 3 3

Corn Starch 397.486 397.486

Maltodextrin 10 132 132

Sucrose 100 100

Cellulose, BW200 100 50

Propionate 0 50

Soybean Oil 70 70

t-Butylhydroquinone 0.014 0.014

Mineral Mix S10022G 35 35

Vitamin Mix V10022G 10 10

Choline Bitartrate 2.5 2.5

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Table S4. Primer sequences used in this study (mouse).

Table S4.

Gene Sequence

18S Forward 5’-CTCAACACGGGAAACCTCAC-3’

Reverse 5’-AGACAAATCGCTCCACCAAC-3’

Gpr41 Forward 5’-GTGACCATGGGGACAAGCTTC-3’

Reverse 5’-CCCTGGCTGTAGGTTGCATT-3’

Gfap Forward 5’-AAGCTCCAAGATGAAACCAACCTGA-3’

Reverse 5’-GCAAACTTAGACCGATACCACTC-3’

Nes Forward 5’-CGCTGGAACAGAGATTGGAAG-3’

Reverse 5’-GTCTCAAGGGTATTAGGCAAG-3’

Th Forward 5’-CGAGGAGAGGGATGGAAATG-3’

Reverse 5’-TCAAAGCCCGAGACAGTGAG-3’

Gpr43 Forward 5’-GGCTTCTACAGCAGCATCTA-3’

Reverse 5’-AAGCACACCAGGAAATTAAG-3’

Pax4 Forward 5’-AAATGGCGCAGGCAAGAGAA-3’

Reverse 5’-ATGAGGAGGAAGCCACAGGA-3’

Pax6 Forward 5’-CCATGTTTGCTTGGGAAATCCG-3’

Reverse 5’-GCTTCATCCGAGTCTTCTCCGTTAG-3’

Nkx6.1 Forward 5’-CTGCACAGTATGGCCGAGATG-3’

Reverse 5’-CCGGGTTATGTGAGCCCAA-3’

Ins2 Forward 5’-CCCTGCTGGCCCTGCTCTT-3’

Reverse 5’-AGGTCTGAAGGTCACCTGCT-3’

Gcg Forward 5’-TGGACTCCCGCCGTGCCCAA-3’

Reverse 5’-CGACTTCTTCTGGGAAGTCTCGCCT-3’

Pyy Forward 5’-CAATGCTGCTAATCCTGCTCG-3’

Reverse 5’-TTTGGAGAACAGAGCTGCGG-3’

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Table S5. Primer sequences used in this study (rat).

Table S5.

Gene Sequence

18S Forward 5’-TTGTTGGTTTTCGGAACTGAG-3’

Reverse 5’-CGGAACTACGACGGTATCTGA-3’

Gpr41 Forward 5’-GCCCTTCGCCATCATC-3’

Reverse 5’-TGCTCCGTGCTGTCTG-3’

Gpr43 Forward 5’-GGCTTCGGCTTCTACAGCAGTATCT-3’

Reverse 5’-TCCAAAGCACACCAGGAAATTAAGA-3’

Ins2 Forward 5’-AGGTTGCCCGGCAGAAG-3’

Reverse 5’-GTTGGTAGAAGGGAGCAGATGCT-3’