Generation of antibody- and B cell-deficient pigs by targeted disruption of the J-region gene segment of the heavy chain locus

Abstract

A poly(A)-trap gene targeting strategy was used to disrupt the single functional heavy chain (HC) joining region (J(H)) of swine in primary fibroblasts. Genetically modified piglets were then generated via somatic cell nuclear transfer (SCNT) and bred to yield litters comprising J(H) wild-type littermate (+/+), J(H) heterozygous knockout (±) and J(H) homozygous knockout (-/-) piglets in the expected Mendelian ratio of 1:2:1. There are only two other targeted loci previously published in swine, and this is the first successful poly(A)-trap strategy ever published in a livestock species. In either blood or secondary lymphoid tissues, flow cytometry, RT-PCR and ELISA detected no circulating IgM(+) B cells, and no transcription or secretion of immunoglobulin (Ig) isotypes, respectively in J(H) -/- pigs. Histochemical and immunohistochemical (IHC) studies failed to detect lymph node (LN) follicles or CD79α(+) B cells, respectively in J(H) -/- pigs. T cell receptor (TCR)(β) transcription and T cells were detected in J(H) -/- pigs. When reared conventionally, J(H) -/- pigs succumbed to bacterial infections after weaning. These antibody (Ab)- and B cell-deficient pigs have significant value as models for both veterinary and human research to discriminate cellular and humoral protective immunity to infectious agents. Thus, these pigs may aid in vaccine development for infectious agents such as the pandemic porcine reproductive and respiratory syndrome virus (PRRSV) and H1N1 swine flu. These pigs are also a first significant step towards generating a pig that expresses fully human, antigen-specific polyclonal Ab to target numerous incurable infectious diseases with high unmet clinical need.

Fig. 1

Targeting strategy for the porcine J_H region. a The top is a schematic of the porcine HC locus containing the J_H region, showing one functional J_H (the dark box most 3′ of the region) and four J_H-like pseudogenes. The middle is an enlarged version of the top focused on the genomic sequence to be targeted with the targeting vector underneath it. The 3′ targeting arm retained the complete J_H to IgM constant region (Cμ) intron, and a few bp of the 3′ J_H coding sequence, to insure normal splicing between the neoR transcript and the poly(A) signals found downstream of the Cμ coding region. Locations of primers used for PCR screening of targeted clones are shown as well (black arrowheads for 5′ and red arrowheads for 3′ end targeting confirmation, respectively). b A representative Southern (S) blot is shown for the nine piglets from the first litter of a J_H ± × J_H ± mating. The Southern probe used for the XbaI digestion is shown as in A. A 22 kb band represents a non-targeted allele (see a). A 3.3 kb band represents a targeted allele (see a). The genotype is given along with the piglet number along the top of the blot

Fig. 2

Expression of IgM on J_H +/+, J_H ± and J_H −/− pigs on WBC. a Histogram of a J_H +/+ piglet (top, piglet # 6), a J_H ± piglet (middle, representative piglet # 4) and a J_H −/− piglet (bottom, representative piglet # 9) after birth. b Shown are data of a J_H +/+ pig (pig # 315-2) in comparison to 3 surviving J_H −/− pigs (pig #s 315-3, 315-4 and 315-9) within a litter reared normally at 8 weeks of age. No J_H −/− pigs have IgM⁺ lymphocytes at either age

Fig. 3

Transcription and serum titers of major Ig isotypes in colostrum-deprived J_H +/+, J_H ± and J_H −/− newborn piglets. a RT-PCR recovery of splenic transcripts encoding rearranged IgA (A) IgG (G) and IgM (M) in piglet piglets 1–9 genotyped in Fig. 1b. Negative controls for the first round PCR (bk 1+2) and the second round (bk 2) are indicated together with a polynucleotide ladder (L). Arrow points out expected band. b RT-PCR recovery of transcripts for IgM from WBC of representative piglets. Arrow points out expected band. c RT-PCR recovery of rearranged TCR_β transcripts from WBC of representative piglets. d Ig levels in the sera of littermates of various genotypes. N/D = below the detection limit of 0.4–0.6 ng/ml; N/A = serum not available for testing. Values are ±Standard Error of the Mean

Fig. 4

Histochemistry and fluorescence IHC of the LNs of J_H +/+, J_H ± and J_H −/− newborn piglets. a MLN were H and E stained and areas of follicular development were analyzed. In a J_H +/+ piglet (top, piglet # 6), follicles were developed and have a distinct germinal center, which is visible at both lower magnification (100×, left side) and at higher magnification (200×, right side, centered on black box in 100×). In one example of a J_H ± piglet (middle, piglet # 1), follicles have structure but are not fully developed, and lack intact germinal centers. In all J_H −/− piglets (bottom, representative piglet # 9), there is no distinguishable follicular or germinal center development. b MLNs were removed and stained with anti-porcine kappa light chain, anti-porcine lambda light chain, anti-porcine IgM, or isotype control Abs. In the J_H +/+ piglet (top, piglet # 6), staining for either light chain and for IgM are positive in MLN follicles. In a J_H ± piglet (middle, representative piglet # 1), a similar staining pattern can be found. In all J_H −/− piglets (bottom, representative piglet # 9), there is no staining for either light chain or IgM. Magnification 200×

Fig. 5

Distribution of T and B cells determined by IHC using anti-CD3 and anti-CD79α in newborn piglets. In a J_H +/+ piglet (top, piglet # 6), follicles were developed and have a distinct germinal center, which is visible at both lower magnification (low, 100×, left side) and at higher magnification (high, 200×, right side, centered on black box in low). In one example of a J_H ± piglet (middle, piglet # 1), follicles have structure but are not fully developed, and lack an intact germinal center. In all J_H −/− piglets (bottom, representative piglet # 9), there is no distinguishable follicular or germinal center development