- Email: [email protected]
Sequence and expression of the FcRn in the porcine mammary gland
Veterinary Immunology and Immunopathology 91 (2003) 227–231
Short communication
Sequence and expression of the FcRn in the porcine mammary gland P.M. Schnulle, W.L. Hurley* Department of Animal Sciences, University of Illinois, Urbana, IL 61801, USA Received 12 September 2002; received in revised form 15 November 2002; accepted 15 November 2002
Abstract Transport of immunoglobulin G across epithelial cell barriers is thought to occur by a system involving the Fcg receptor called the neonatal Fc receptor (FcRn). The FcRn may also play a role in IgG transport in the mammary gland. To determine the presence of FcRn in the porcine mammary gland, biopsies were taken from glands 3 days prepartum and on the day of farrowing. The full length porcine FcRn cDNA sequence was obtained by rapid amplification of cDNA ends and determined to be 1557 base pairs in length that codes for a 359 amino acid peptide. Expression of FcRn mRNA in the porcine mammary gland was determined by reverse transcriptase-PCR and revealed that the mRNA is present prepartum and on the day of farrowing. These results indicate that the FcRn is expressed in porcine mammary tissue and are consistent with the hypothesis that FcRn may have a role in mammary gland immunoglobulin transport during colostrogenesis. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Neonatal Fc receptor; IgG transport; Colostrum; Pig; Mammary gland
1. Introduction Colostrum is important for the survival of the porcine neonate. Piglets are thought to be born agammaglobulinemic, although recent studies show that some maternal IgG may be crossing the placenta into the fetus (Butler et al., 2002). However, piglets still require the immunoglobulins from colostrum to obtain Abbreviations: cDNA, complementary deoxyribonucleic acid; CIP, calf intestinal phosphatase; DNA, deoxyribonucleic acid; dNTP, deoxynucleotide triphosphates; FcRn, neonatal Fc receptor; M-MLV RT, moloney-murine leukemia virus reverse transcriptase; mRNA, messenger ribonucleic acid; RACE, rapid amplification of cDNA ends; RNA, ribonucleic acid; RT, reverse transcriptase * Corresponding author. Tel.: þ1-217-333-1327; fax: þ1-217-333-8804. E-mail address: [email protected] (W.L. Hurley).
sufficient immunity during early postnatal development. Absorption of immunoglobulins by the piglet occurs over a brief period of approximately 36 h postpartum. In the mammary gland, immunoglobulins are selectively transported across the epithelial cells into the lumen of the alveolus by a receptor-mediated process. In the pig, IgG is preferentially transported during colostrum formation, however, the specific receptor involved in IgG transport in the mammary gland is unknown. The immunoglobulin Fcg receptor FcRn was originally described as the IgG transporter in the gut of the neonatal rat (Simister and Mostov, 1989). Since then, it has been studied as a possible IgG transporter in the mammary gland. The FcRn has been localized in the lactating mammary gland of the bovine (Kacskovics et al., 2000), mouse (Cianga et al., 1999),
0165-2427/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 0 2 ) 0 0 2 9 4 - 5
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P.M. Schnulle, W.L. Hurley / Veterinary Immunology and Immunopathology 91 (2003) 227–231
brushtail possum (Adamski et al., 2000), and sheep (Mayer et al., 2002) indicating a potential association with transepithelial IgG transport. Some research suggests that the FcRn may be serving as a recycling mechanism for IgG in the lactating mammary gland of the mouse (Cianga et al., 1999). More recently, localization of FcRn in the porcine placenta has been reported (Butler et al., 2002), although the specific data remains unpublished at this time. The objective of the present study was to identify FcRn in the pig mammary gland and determine the presence of FcRn mRNA during the peripartum period.
2. Materials and methods Mammary punch biopsies were performed to obtain tissue from six Yorkshire pigs approximately 3 days prepartum and within 24 h after farrowing. Biopsies were performed by first sedating the sow with Acepromazine Maleate (Boeheringer Ingelheim Vetmedica Inc., St. Joseph, MO) at a dose of 0.2–0.5 mg/kg i.m. Lidocaine HCl (2.5–3.0 ml of a 2% solution; Abbott Laboratories, North Chicago, IL) was administered directly under the skin of the mammary gland. A small incision was made into the gland and biopsies were collected using a MAGNUM1 biopsy instrument fitted with a sterile MAGNUM1 core tissue biopsy needle (C.R. Bard Inc., Covington, GA). Incisions were closed with a single suture. Sows were injected i.m. with 3 ml of FlunixaminTM (Fort Dodge Laboratories, Overland Park, KS) to alleviate inflammation. Total RNA was isolated from mammary samples using UltraspecTM-II RNA Isolation kit (Biotecx Laboratories Inc., Houston, TX) following the manufacturer’s instructions. Pooled ribonucleic acid (RNA) from the day of farrowing from three pigs was used to obtain the full sequence of the FcRn using rapid amplification of cDNA ends (First ChoiceTM RLM-RACE Kit, Ambion, Austin, TX). All materials and protocols used were provided with the kit unless otherwise stated. To obtain the 50 end, 10 mg of pooled RNA were used. The RNA was first processed according to the protocol provided with the kit by treating with 2 ml of calf intestinal phosphatase (CIP) and 2 ml of 10 CIP buffer in a total volume 20 ml and incubated for 1 h at 37 8C.
To terminate the CIP reaction, 15 ml of ammonium acetate solution, 115 ml of water and 150 ml of acid phenol: chloroform (Ambion, Austin, TX) were added. The reaction was centrifuged for 5 min at 10,000 g at room temperature. The aqueous phase was transferred to a new tube to which 150 ml of chloroform (Fisher Scientific, Fair Lawn, NJ) were added. The sample was mixed by vortexing and centrifuged for 5 min at room temperature at 10,000 g. The aqueous phase was transferred to a new tube to which 150 ml of isopropanol (EM Science, Gibbstown, NJ) were added. The sample was vortexed and then stored on ice for 10 min. After chilling, the sample was centrifuged at 13,000 g for 20 min. The supernatant was discarded and the remaining pellet was rinsed with 500 ml of cold 70% ethanol. The tube was centrifuged for 5 min at 13,000 g speed, the ethanol carefully removed and the pellet was allowed to air dry. The pellet was resuspended in 11 ml of water. Two microliters of the CIP-treated RNA was added to 2 ml of tobacco acid pyrophosphatase, 1 ml of 10 tobacco acid pyrophosphatase buffer and 2 ml of water. The sample was incubated for 1 h at 37 8C. Two microliters of the tobacco acid pyrophosphatase reaction were then added to 1 ml of 50 RACE Adapter, 1 ml of 10 RNA Ligase buffer, 2 ml of T4 RNA Ligase and 4 ml of water. The reaction was incubated at 37 8C for 1 h to allow for ligation of the adapter. The reverse transcriptase (RT) reaction also was performed according to the protocol provided with the First ChoiceTM RLM-RACE Kit. Two microliters of ligated RNA were mixed with 4 ml of deoxynucleotide triphosphate (dNTP) mix, 2 ml of Random Decamers, 2 ml of 10 RT buffer, 1 ml of RNase inhibitor and 1 ml of moloney-murine leukemia virus reverse transcriptase (M-MLV RT) to a final volume of 20 ml. The RT reaction was incubated in a PTC-100 thermal cycler (MJ Research, Watertown, MA) at 42 8C for 1 h and maintained at 4 8C. Nested primers, used to obtain the 50 end, were designed from a multiple sequence alignment of complete cDNA sequences from the bovine (Kacskovics et al., 2000), human (Story et al., 1994), mouse (Ahouse et al., 1993) and rat (Simister and Mostov, 1989) and from a partial ovine sequence (accession no. AJ313190): F1R (50 -CAGGTGGGTAGAAGGAGAAG-30 ) and F2R (50 -GGCTCCTTCCACTCCAGGTT-30 ). All primers were made by Operon (Alameda, CA).
P.M. Schnulle, W.L. Hurley / Veterinary Immunology and Immunopathology 91 (2003) 227–231
The 50 end was obtained following the protocol provided with the kit with the addition of 50 mg/ml of BSA (Kreader, 1996) to the outer nested PCR reaction and using 1.25 units Super Taq Plus Polymerase (Ambion, Austin, TX) for the outer reaction and 1 bead of TaqBeadTM Hot Start Polymerase (Promega, Madison, WI) for the inner reaction. Both the outer and inner PCR reactions were carried out in a PTC-100 thermal cycler (MJ Research, Watertown, MA) under the following conditions: (i) 94 8C for 3 min; (ii) 35 cycles of 94 8C for 1 min, 56 8C for 1 min, 72 8C for 1.5 min; (iii) 72 8C for 7 min; (iv) maintenance of reaction at 4 8C. The PCR product was purified using the Qiaquick PCR Purification Kit (Qiagen, Valencia, CA). The purified PCR product was sequenced by the HighThroughput Sequencing Unit of the W.M. Keck Center for Comparative and Functional Genomics in the Biotechnology Center at the University of Illinois Urbana-Champaign. Sequencing was done in an ABI PRISM1 377 96 lane automated DNA sequencer (Foster City, CA). To obtain the 30 end sequence, 1 mg of pooled RNA was reverse transcribed by adding 4 ml of dNTP mix, 2 ml of 30 RACE adapter, 2 ml of 10 RT buffer, 1 ml of RNase inhibitor, 1 ml of M-MLV RT and 8 ml of water, and incubating for 1 h at 42 8C. From the 50 end sequence, two nested primers were designed to obtain the 30 end: F1F (50 -CTCTCAAAACCTTGGAGGAA30 ) and F2F (50 -ATGAAGTTCGACACCAAGCT-30 ). The 30 end nested PCR was done following the company’s protocol with the addition of 50 mg/ml of BSA (Kreader, 1996) to the outer nested PCR reaction and using 1 bead of TaqBeadTM Hot Start Polymerase (Promega, Madison, WI) for each outer and inner PCR reaction. The 30 PCR reaction was carried out and purified in the same way as for the 50 PCR reaction. The PCR product was sequenced as stated previously. An additional forward primer was required to obtain the extreme 30 end sequence of the 30 end: F3F (50 -GGCTTCCTACTGCTCTTGAT-30 ). This primer was used only in the sequencing reaction. To profile FcRn expression in the mammary gland using RT-PCR, 2.0 mg of total RNA were first denatured at 75 8C for 3 min. Added to the RNA was a mixture containing 0.9 mM of each dNTP (Promega, Madison, WI), 50 units of Recombinant RNasin1 Ribonuclease Inhibitor (Promega, Madison, WI), 10 ml of M-MLV RT 5 Buffer (Promega, Madison,
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Fig. 1. Coding region sequence comparison of the FcRn from the porcine, bovine, human and rat. Nucleotide homology is indicated by a star. Gaps in sequence alignment are indicated by a dash. Start and stop codons of all species are underlined. The boxed codon encodes a single alanine that is present only in the porcine FcRn.
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WI), 0.05 mg of BSA (Promega, Madison, WI), 0.5 mg of Oligo(dT)15 Primer (Promega, Madison, WI), 1.8 mM DTT (Life Technologies Inc., Rockville, MD), and 400 units of M-MLV RT (Promega, Madison, WI) in a final volume of 55 ml. The RT reaction was carried out in a PTC-100 Thermal Cycler under the following conditions: (i) 42 8C for 1 h; (ii) 72 8C for 10 min; (iii) maintenance of the reaction at 4 8C. Six microliters of cDNA were used for each PCR reaction. The PCR reaction mixture contained 0.2 mM of each dNTP, 10% 10 Reaction Buffer (Promega, Madison, WI), 3.0 mM MgCl2 (Promega, Madison, WI), 0.1 mM forward primer, F2F (50 -ATGAAGTTCGACACCAAGCT-30 ), 0.1 mM reverse primer, F1R (50 -CAGGTGGGTAGAAGGAGAAG-30 ), and 1 bead of TaqBeadTM Hot Start Polymerase in a final volume of 100 ml per reaction. The PCR reactions were performed in a PTC-100 thermal cycler under the following conditions: (i) 94 8C for 5 min; (ii) 30 cycles of 94 8C for 1 min, 50 8C for 1 min, 72 8C for 1 min; (iii) 72 8C for 10 min; (iv) maintenance of reaction at 4 8C. To visualize the PCR products, 30 ml of each reaction with 3 ml of loading dye (Promega, Madison, WI) were run on 1% ultraPURETM agarose (Bethesda Research Laboratories, Gaithersburg, MD) gels with 0.2% ethidium bromide (Sigma, St. Louis, MO) in 1 TAE (40 mM trizma base, 20 mM sodium acetate, and 1 mM EDTA). Gels were visualized using a FOTO/ UV1 26 transilluminator (Fotodyne Incorporated, Hartland, WI) and photographs taken with a video camera using the NIH Imager program.
3. Results and discussion Sequencing of the RACE products produced a complete cDNA sequence of 1557 base pairs (Genbank
accession no. AY135635) with a 60% GC content. The porcine FcRn is highly homologous with the bovine sequence sharing 85% identity in the coding region (Fig. 1) and 85% identity overall. The porcine FcRn cDNA coding region is also 82 and 69% identical with the coding regions of the human and rat, respectively (Fig. 1). The overall porcine sequence is 81, 69 and 70% identical with the FcRn sequence in the human, rat and mouse, respectively. The porcine FcRn cDNA sequence is shorter than the rat, human and mouse sequences. A protein sequence of 359 amino acids was derived from the cDNA sequence (Genbank protein ID AAN23851). This protein sequence of the porcine FcRn also has high homology with the bovine protein sequence, sharing 84% identity. The porcine protein sequence is 68, 76 and 66% identical with the mouse, human and rat, respectively. The porcine sequence has an additional alanine not present in the bovine, human, mouse or rat (boxed codon in Fig. 1). The derived porcine protein sequence is longer than the bovine sequence but is shorter than the human, mouse or rat sequences. Profiling of the expression of FcRn mRNA in the porcine mammary gland during the prepartum period (approximately 3 days prefarrowing) and immediately postfarrowing revealed that the FcRn is present during both periods (Fig. 2). Although there is some variability among sows, there may be a general decrease in expression on the day of farrowing relative to the prepartum period (Fig. 2). Further studies are required to determine the quantitative differences in FcRn expression. Recently, it was reported that the FcRn can be localized in the porcine placenta (Butler et al., 2002), although this research is unpublished at this time. Butler et al. (2002) have hypothesized that the piglet may be born with a low level of serum IgG due
Fig. 2. Expression of porcine FcRn mRNA in the mammary gland. The expression of FcRn mRNA was determined by RT-PCR using RNA from three sows. The FcRn mRNA was expressed at detectable levels at approximately 3 days prepartum (P) in all the three sows and on the day of farrowing (F) in two of the sows. Reaction loading control was RT-PCR of 18S ribosomal RNA.
P.M. Schnulle, W.L. Hurley / Veterinary Immunology and Immunopathology 91 (2003) 227–231
to selective transport across the placenta. Nevertheless, colostrum is still the major source of IgG for passive immunity in the porcine neonate. Serum IgG levels decrease 3 days prepartum in the sow (Huang et al., 1992) and IgG is preferentially transported in the mammary gland during colostrogenesis (Watson, 1980). It is also known that the FcRn binds IgG (Simister and Mostov, 1989). An increase in FcRn mRNA expression in the pig around the time of parturition would be consistent with the hypothesis that the porcine FcRn is associated with IgG transport in the mammary gland. However, recent studies in the mouse also indicate a potential role for the FcRn as a recycler of IgG in the mammary tissue (Cianga et al., 1999).
4. Conclusions This present study confirms that FcRn is expressed in the sow’s mammary gland. The expression of FcRn occurs during the time of colostrogenesis. Further study is required to confirm that the FcRn expressed in the pig mammary gland is responsible for the extensive IgG transport occurring during colostrogenesis. Acknowledgements The authors wish to acknowledge the generous help and valuable instruction of Dr. M. Noble. This material is based on work partially supported by the Illinois Agricultural Experimental Station as part of Animal Health and Disease Formula Project 35-0975 and Hatch Project 35-0344.
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References Adamski, F.M., King, A.T., Demmer, J., 2000. Expression of the Fc receptor in the mammary gland during lactation in the marsupial Trichosurus vulpecula (brushtail possum). Mol. Immunol. 37, 435–444. Ahouse, J.J., Hagerman, C.L., Mittal, P., Gilbert, D.J., Copeland, N.G., Jenkins, N.A., Simister, N.E., 1993. Mouse MHC class Ilike Fc receptor encoded outside the MHC. J. Immunol. 151, 6078–6088. Butler, J.E., Sun, J., Weber, P., Ford, S.P., Rehakova, Z., Sinkora, J., Franscis, D., Lager, K., 2002. Switch recombination in fetal porcine thymus is uncoupled from somatic mutation. Vet. Immunol. 87, 307–319. Cianga, P., Corneliu, M., Richardson, J.A., Ghetie, V., Ward, E.S., 1999. Identification and function of neonatal Fc receptor in mammary gland of lactating mice. Eur. J. Immunol. 29, 2515– 2523. Huang, S., Hu, Z., Hasler-Rapacz, J., Rapacz, J., 1992. Preferential mammary storage and secretion of immunoglobulin gamma (IgG) subclasses in swine. J. Reprod. Immunol. 21, 15–28. Kacskovics, I., Wu, Z., Simister, N.E., Frenyo´ , L.V., Hammarstro¨ m, L., 2000. Cloning and characterization of the bovine MHC class I-like Fc receptor. J. Immunol. 164, 1889–1897. Kreader, C.A., 1996. Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein. Appl. Environ. Microbiol. 62, 1102–1106. Mayer, B., Zolnai, A., Frenyo´ , L.V., Jancsik, V., Szentirmay, Z., Hammarstro¨ m, L., Kacskovics, I., 2002. Localization of the sheep FcRn in the mammary gland. Vet. Immunol. 87, 327– 330. Simister, N.E., Mostov, K.E., 1989. An Fc receptor structurally related to MHC class I antigens. Nature 337, 184–187. Story, C.M., Mikulska, J., Simister, N.E., 1994. A major histocompatibility complex class I-like Fc receptor cloned from human placenta: possible role in transfer of immunoglobulin G from mother to fetus. J. Exp. Med. 180, 2377–2381. Watson, D.L., 1980. Immunological functions of the mammary gland and its secretion: comparative review. Aust. J. Biol. Sci. 33, 403–422.
Short communication
Sequence and expression of the FcRn in the porcine mammary gland P.M. Schnulle, W.L. Hurley* Department of Animal Sciences, University of Illinois, Urbana, IL 61801, USA Received 12 September 2002; received in revised form 15 November 2002; accepted 15 November 2002
Abstract Transport of immunoglobulin G across epithelial cell barriers is thought to occur by a system involving the Fcg receptor called the neonatal Fc receptor (FcRn). The FcRn may also play a role in IgG transport in the mammary gland. To determine the presence of FcRn in the porcine mammary gland, biopsies were taken from glands 3 days prepartum and on the day of farrowing. The full length porcine FcRn cDNA sequence was obtained by rapid amplification of cDNA ends and determined to be 1557 base pairs in length that codes for a 359 amino acid peptide. Expression of FcRn mRNA in the porcine mammary gland was determined by reverse transcriptase-PCR and revealed that the mRNA is present prepartum and on the day of farrowing. These results indicate that the FcRn is expressed in porcine mammary tissue and are consistent with the hypothesis that FcRn may have a role in mammary gland immunoglobulin transport during colostrogenesis. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Neonatal Fc receptor; IgG transport; Colostrum; Pig; Mammary gland
1. Introduction Colostrum is important for the survival of the porcine neonate. Piglets are thought to be born agammaglobulinemic, although recent studies show that some maternal IgG may be crossing the placenta into the fetus (Butler et al., 2002). However, piglets still require the immunoglobulins from colostrum to obtain Abbreviations: cDNA, complementary deoxyribonucleic acid; CIP, calf intestinal phosphatase; DNA, deoxyribonucleic acid; dNTP, deoxynucleotide triphosphates; FcRn, neonatal Fc receptor; M-MLV RT, moloney-murine leukemia virus reverse transcriptase; mRNA, messenger ribonucleic acid; RACE, rapid amplification of cDNA ends; RNA, ribonucleic acid; RT, reverse transcriptase * Corresponding author. Tel.: þ1-217-333-1327; fax: þ1-217-333-8804. E-mail address: [email protected] (W.L. Hurley).
sufficient immunity during early postnatal development. Absorption of immunoglobulins by the piglet occurs over a brief period of approximately 36 h postpartum. In the mammary gland, immunoglobulins are selectively transported across the epithelial cells into the lumen of the alveolus by a receptor-mediated process. In the pig, IgG is preferentially transported during colostrum formation, however, the specific receptor involved in IgG transport in the mammary gland is unknown. The immunoglobulin Fcg receptor FcRn was originally described as the IgG transporter in the gut of the neonatal rat (Simister and Mostov, 1989). Since then, it has been studied as a possible IgG transporter in the mammary gland. The FcRn has been localized in the lactating mammary gland of the bovine (Kacskovics et al., 2000), mouse (Cianga et al., 1999),
0165-2427/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 0 2 ) 0 0 2 9 4 - 5
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brushtail possum (Adamski et al., 2000), and sheep (Mayer et al., 2002) indicating a potential association with transepithelial IgG transport. Some research suggests that the FcRn may be serving as a recycling mechanism for IgG in the lactating mammary gland of the mouse (Cianga et al., 1999). More recently, localization of FcRn in the porcine placenta has been reported (Butler et al., 2002), although the specific data remains unpublished at this time. The objective of the present study was to identify FcRn in the pig mammary gland and determine the presence of FcRn mRNA during the peripartum period.
2. Materials and methods Mammary punch biopsies were performed to obtain tissue from six Yorkshire pigs approximately 3 days prepartum and within 24 h after farrowing. Biopsies were performed by first sedating the sow with Acepromazine Maleate (Boeheringer Ingelheim Vetmedica Inc., St. Joseph, MO) at a dose of 0.2–0.5 mg/kg i.m. Lidocaine HCl (2.5–3.0 ml of a 2% solution; Abbott Laboratories, North Chicago, IL) was administered directly under the skin of the mammary gland. A small incision was made into the gland and biopsies were collected using a MAGNUM1 biopsy instrument fitted with a sterile MAGNUM1 core tissue biopsy needle (C.R. Bard Inc., Covington, GA). Incisions were closed with a single suture. Sows were injected i.m. with 3 ml of FlunixaminTM (Fort Dodge Laboratories, Overland Park, KS) to alleviate inflammation. Total RNA was isolated from mammary samples using UltraspecTM-II RNA Isolation kit (Biotecx Laboratories Inc., Houston, TX) following the manufacturer’s instructions. Pooled ribonucleic acid (RNA) from the day of farrowing from three pigs was used to obtain the full sequence of the FcRn using rapid amplification of cDNA ends (First ChoiceTM RLM-RACE Kit, Ambion, Austin, TX). All materials and protocols used were provided with the kit unless otherwise stated. To obtain the 50 end, 10 mg of pooled RNA were used. The RNA was first processed according to the protocol provided with the kit by treating with 2 ml of calf intestinal phosphatase (CIP) and 2 ml of 10 CIP buffer in a total volume 20 ml and incubated for 1 h at 37 8C.
To terminate the CIP reaction, 15 ml of ammonium acetate solution, 115 ml of water and 150 ml of acid phenol: chloroform (Ambion, Austin, TX) were added. The reaction was centrifuged for 5 min at 10,000 g at room temperature. The aqueous phase was transferred to a new tube to which 150 ml of chloroform (Fisher Scientific, Fair Lawn, NJ) were added. The sample was mixed by vortexing and centrifuged for 5 min at room temperature at 10,000 g. The aqueous phase was transferred to a new tube to which 150 ml of isopropanol (EM Science, Gibbstown, NJ) were added. The sample was vortexed and then stored on ice for 10 min. After chilling, the sample was centrifuged at 13,000 g for 20 min. The supernatant was discarded and the remaining pellet was rinsed with 500 ml of cold 70% ethanol. The tube was centrifuged for 5 min at 13,000 g speed, the ethanol carefully removed and the pellet was allowed to air dry. The pellet was resuspended in 11 ml of water. Two microliters of the CIP-treated RNA was added to 2 ml of tobacco acid pyrophosphatase, 1 ml of 10 tobacco acid pyrophosphatase buffer and 2 ml of water. The sample was incubated for 1 h at 37 8C. Two microliters of the tobacco acid pyrophosphatase reaction were then added to 1 ml of 50 RACE Adapter, 1 ml of 10 RNA Ligase buffer, 2 ml of T4 RNA Ligase and 4 ml of water. The reaction was incubated at 37 8C for 1 h to allow for ligation of the adapter. The reverse transcriptase (RT) reaction also was performed according to the protocol provided with the First ChoiceTM RLM-RACE Kit. Two microliters of ligated RNA were mixed with 4 ml of deoxynucleotide triphosphate (dNTP) mix, 2 ml of Random Decamers, 2 ml of 10 RT buffer, 1 ml of RNase inhibitor and 1 ml of moloney-murine leukemia virus reverse transcriptase (M-MLV RT) to a final volume of 20 ml. The RT reaction was incubated in a PTC-100 thermal cycler (MJ Research, Watertown, MA) at 42 8C for 1 h and maintained at 4 8C. Nested primers, used to obtain the 50 end, were designed from a multiple sequence alignment of complete cDNA sequences from the bovine (Kacskovics et al., 2000), human (Story et al., 1994), mouse (Ahouse et al., 1993) and rat (Simister and Mostov, 1989) and from a partial ovine sequence (accession no. AJ313190): F1R (50 -CAGGTGGGTAGAAGGAGAAG-30 ) and F2R (50 -GGCTCCTTCCACTCCAGGTT-30 ). All primers were made by Operon (Alameda, CA).
P.M. Schnulle, W.L. Hurley / Veterinary Immunology and Immunopathology 91 (2003) 227–231
The 50 end was obtained following the protocol provided with the kit with the addition of 50 mg/ml of BSA (Kreader, 1996) to the outer nested PCR reaction and using 1.25 units Super Taq Plus Polymerase (Ambion, Austin, TX) for the outer reaction and 1 bead of TaqBeadTM Hot Start Polymerase (Promega, Madison, WI) for the inner reaction. Both the outer and inner PCR reactions were carried out in a PTC-100 thermal cycler (MJ Research, Watertown, MA) under the following conditions: (i) 94 8C for 3 min; (ii) 35 cycles of 94 8C for 1 min, 56 8C for 1 min, 72 8C for 1.5 min; (iii) 72 8C for 7 min; (iv) maintenance of reaction at 4 8C. The PCR product was purified using the Qiaquick PCR Purification Kit (Qiagen, Valencia, CA). The purified PCR product was sequenced by the HighThroughput Sequencing Unit of the W.M. Keck Center for Comparative and Functional Genomics in the Biotechnology Center at the University of Illinois Urbana-Champaign. Sequencing was done in an ABI PRISM1 377 96 lane automated DNA sequencer (Foster City, CA). To obtain the 30 end sequence, 1 mg of pooled RNA was reverse transcribed by adding 4 ml of dNTP mix, 2 ml of 30 RACE adapter, 2 ml of 10 RT buffer, 1 ml of RNase inhibitor, 1 ml of M-MLV RT and 8 ml of water, and incubating for 1 h at 42 8C. From the 50 end sequence, two nested primers were designed to obtain the 30 end: F1F (50 -CTCTCAAAACCTTGGAGGAA30 ) and F2F (50 -ATGAAGTTCGACACCAAGCT-30 ). The 30 end nested PCR was done following the company’s protocol with the addition of 50 mg/ml of BSA (Kreader, 1996) to the outer nested PCR reaction and using 1 bead of TaqBeadTM Hot Start Polymerase (Promega, Madison, WI) for each outer and inner PCR reaction. The 30 PCR reaction was carried out and purified in the same way as for the 50 PCR reaction. The PCR product was sequenced as stated previously. An additional forward primer was required to obtain the extreme 30 end sequence of the 30 end: F3F (50 -GGCTTCCTACTGCTCTTGAT-30 ). This primer was used only in the sequencing reaction. To profile FcRn expression in the mammary gland using RT-PCR, 2.0 mg of total RNA were first denatured at 75 8C for 3 min. Added to the RNA was a mixture containing 0.9 mM of each dNTP (Promega, Madison, WI), 50 units of Recombinant RNasin1 Ribonuclease Inhibitor (Promega, Madison, WI), 10 ml of M-MLV RT 5 Buffer (Promega, Madison,
229
Fig. 1. Coding region sequence comparison of the FcRn from the porcine, bovine, human and rat. Nucleotide homology is indicated by a star. Gaps in sequence alignment are indicated by a dash. Start and stop codons of all species are underlined. The boxed codon encodes a single alanine that is present only in the porcine FcRn.
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WI), 0.05 mg of BSA (Promega, Madison, WI), 0.5 mg of Oligo(dT)15 Primer (Promega, Madison, WI), 1.8 mM DTT (Life Technologies Inc., Rockville, MD), and 400 units of M-MLV RT (Promega, Madison, WI) in a final volume of 55 ml. The RT reaction was carried out in a PTC-100 Thermal Cycler under the following conditions: (i) 42 8C for 1 h; (ii) 72 8C for 10 min; (iii) maintenance of the reaction at 4 8C. Six microliters of cDNA were used for each PCR reaction. The PCR reaction mixture contained 0.2 mM of each dNTP, 10% 10 Reaction Buffer (Promega, Madison, WI), 3.0 mM MgCl2 (Promega, Madison, WI), 0.1 mM forward primer, F2F (50 -ATGAAGTTCGACACCAAGCT-30 ), 0.1 mM reverse primer, F1R (50 -CAGGTGGGTAGAAGGAGAAG-30 ), and 1 bead of TaqBeadTM Hot Start Polymerase in a final volume of 100 ml per reaction. The PCR reactions were performed in a PTC-100 thermal cycler under the following conditions: (i) 94 8C for 5 min; (ii) 30 cycles of 94 8C for 1 min, 50 8C for 1 min, 72 8C for 1 min; (iii) 72 8C for 10 min; (iv) maintenance of reaction at 4 8C. To visualize the PCR products, 30 ml of each reaction with 3 ml of loading dye (Promega, Madison, WI) were run on 1% ultraPURETM agarose (Bethesda Research Laboratories, Gaithersburg, MD) gels with 0.2% ethidium bromide (Sigma, St. Louis, MO) in 1 TAE (40 mM trizma base, 20 mM sodium acetate, and 1 mM EDTA). Gels were visualized using a FOTO/ UV1 26 transilluminator (Fotodyne Incorporated, Hartland, WI) and photographs taken with a video camera using the NIH Imager program.
3. Results and discussion Sequencing of the RACE products produced a complete cDNA sequence of 1557 base pairs (Genbank
accession no. AY135635) with a 60% GC content. The porcine FcRn is highly homologous with the bovine sequence sharing 85% identity in the coding region (Fig. 1) and 85% identity overall. The porcine FcRn cDNA coding region is also 82 and 69% identical with the coding regions of the human and rat, respectively (Fig. 1). The overall porcine sequence is 81, 69 and 70% identical with the FcRn sequence in the human, rat and mouse, respectively. The porcine FcRn cDNA sequence is shorter than the rat, human and mouse sequences. A protein sequence of 359 amino acids was derived from the cDNA sequence (Genbank protein ID AAN23851). This protein sequence of the porcine FcRn also has high homology with the bovine protein sequence, sharing 84% identity. The porcine protein sequence is 68, 76 and 66% identical with the mouse, human and rat, respectively. The porcine sequence has an additional alanine not present in the bovine, human, mouse or rat (boxed codon in Fig. 1). The derived porcine protein sequence is longer than the bovine sequence but is shorter than the human, mouse or rat sequences. Profiling of the expression of FcRn mRNA in the porcine mammary gland during the prepartum period (approximately 3 days prefarrowing) and immediately postfarrowing revealed that the FcRn is present during both periods (Fig. 2). Although there is some variability among sows, there may be a general decrease in expression on the day of farrowing relative to the prepartum period (Fig. 2). Further studies are required to determine the quantitative differences in FcRn expression. Recently, it was reported that the FcRn can be localized in the porcine placenta (Butler et al., 2002), although this research is unpublished at this time. Butler et al. (2002) have hypothesized that the piglet may be born with a low level of serum IgG due
Fig. 2. Expression of porcine FcRn mRNA in the mammary gland. The expression of FcRn mRNA was determined by RT-PCR using RNA from three sows. The FcRn mRNA was expressed at detectable levels at approximately 3 days prepartum (P) in all the three sows and on the day of farrowing (F) in two of the sows. Reaction loading control was RT-PCR of 18S ribosomal RNA.
P.M. Schnulle, W.L. Hurley / Veterinary Immunology and Immunopathology 91 (2003) 227–231
to selective transport across the placenta. Nevertheless, colostrum is still the major source of IgG for passive immunity in the porcine neonate. Serum IgG levels decrease 3 days prepartum in the sow (Huang et al., 1992) and IgG is preferentially transported in the mammary gland during colostrogenesis (Watson, 1980). It is also known that the FcRn binds IgG (Simister and Mostov, 1989). An increase in FcRn mRNA expression in the pig around the time of parturition would be consistent with the hypothesis that the porcine FcRn is associated with IgG transport in the mammary gland. However, recent studies in the mouse also indicate a potential role for the FcRn as a recycler of IgG in the mammary tissue (Cianga et al., 1999).
4. Conclusions This present study confirms that FcRn is expressed in the sow’s mammary gland. The expression of FcRn occurs during the time of colostrogenesis. Further study is required to confirm that the FcRn expressed in the pig mammary gland is responsible for the extensive IgG transport occurring during colostrogenesis. Acknowledgements The authors wish to acknowledge the generous help and valuable instruction of Dr. M. Noble. This material is based on work partially supported by the Illinois Agricultural Experimental Station as part of Animal Health and Disease Formula Project 35-0975 and Hatch Project 35-0344.
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