New Wistar Kyoto and Spontaneously Hypertensive rat transgenic models with ubiquitous expression of green fluorescent protein

The Wistar Kyoto (WKY) rat and the spontaneously hypertensive (SHR) rat inbred strains are well-established models for human crescentic glomerulonephritis (CRGN) and metabolic syndrome, respectively. Novel transgenic (Tg) strains add research opportunities and increase scientific value to well-established rat models. We have created two novel Tg strains using Sleeping Beauty transposon germline transgenesis, ubiquitously expressing green fluorescent protein (GFP) under the rat elongation factor 1 alpha (EF1a) promoter on the WKY and SHR genetic backgrounds. The Sleeping Beauty system functioned with high transgenesis efficiency; 75% of new rats born after embryo microinjections were transgene positive. By ligation-mediated PCR, we located the genome integration sites, confirming no exonic disruption and defining a single or low copy number of the transgenes in the new WKY-GFP and SHR-GFP Tg lines. We report GFP-bright expression in embryos, tissues and organs in both lines and show preliminary in vitro and in vivo imaging data that demonstrate the utility of the new GFP-expressing lines for adoptive transfer, transplantation and fate mapping studies of CRGN, metabolic syndrome and other traits for which these strains have been extensively studied over the past four decades

Compensatory T-Cell Regulation in Unaffected Relatives of SLE Patients, and Opposite IL-2/CD25-Mediated Effects Suggested by Coreferentiality Modeling

In human systemic lupus erythematosus (SLE), diverse autoantibodies accumulate over years before disease manifestation. Unaffected relatives of SLE patients frequently share a sustained production of autoantibodies with indiscriminable specificity, usually without ever acquiring the disease. We studied relations of IgG autoantibody profiles and peripheral blood activated regulatory T-cells (aTregs), represented by CD4+CD25bright T-cells that were regularly 70–90% Foxp3+. We found consistent positive correlations of broad-range as well as specific SLE-associated IgG with aTreg frequencies within unaffected relatives, but not patients or unrelated controls. Our interpretation: unaffected relatives with shared genetic factors compensated pathogenic effects by aTregs engaged in parallel with the individual autoantibody production. To study this further, we applied a novel analytic approach named coreferentiality that tests the indirect relatedness of parameters in respect to multivariate phenotype data. Results show that independently of their direct correlation, aTreg frequencies and specific SLE-associated IgG were likely functionally related in unaffected relatives: they significantly parallelled each other in their relations to broad-range immunoblot autoantibody profiles. In unaffected relatives, we also found coreferential effects of genetic variation in the loci encoding IL-2 and CD25. A model of CD25 functional genetic effects constructed by coreferentiality maximization suggests that IL-2-CD25 interaction, likely stimulating aTregs in unaffected relatives, had an opposed effect in SLE patients, presumably triggering primarily T-effector cells in this group. Coreferentiality modeling as we do it here could also be useful in other contexts, particularly to explore combined functional genetic effects

Coreferentialities with CD25^bright/CD4⁺ frequencies.

*<p>Significance according to permutation test (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033992#s4" target="_blank">methods</a>). Effects of bystander coreferentiality were checked for all significant tests (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033992#s4" target="_blank">methods</a>), but rates of simulated bystander data reaching the respective test significance never exceeded 5%.</p

Unweighted and family-weighted correlation coefficients.

<p>Distributions of unweighted and family-wighted correlation coefficients of CD25^bright/CD4⁺ aTreg frequencies (A) and log-transformed IgG anti-Sm (B) with all 130 single immunoblot reactivities are shown within SLE patients, unaffected relatives and unrelated controls, respectively. Reactivities to cytoplasmic bands are indicated by filled circles, anti-nuclear reactivities by triangles and anti-brain reactivities by crosses. Annoteted P-values were calculated by the described permutation test.</p

IgG autoreactivities in SLE patients, unafffected relatives and unrelated control subjects.

<p>A–F: Band numbers and 1st principal component calculated from HEp2 anti–cytoplasmic (A,B), anti-nuclear (C,D) and anti-brain (E,F) imunoblot reactivities. G–J: Specific SLE-associated autoreactive IgG quantified by ELISA. All plots show group-wise medians and results of pairwise Mann-Whitney tests for differences between groups.</p

Group-wise coreferentialities between CD25^bright/CD4+ aTreg frequencies and <i>IL2RA</i> genetic-effects model scores.

<p>A: SLE patients, B: unaffected relatives, C: unrelated controls. Reactivities to cytoplasmic bands are indicated by filled circles, anti-nuclear reactivities by triangles and anti-brain reactivities by crosses.</p

Coreferentialities of the <i>IL2RA</i> model score with other parameters.

*<p>Test for multiple parameters, on maximal absolute coreferentiality |<i>R_C</i>|_max (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033992#s4" target="_blank">methods</a>). Significant tests, except those for multiple SNPs, were also checked for the effect of bystander coreferentiality (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033992#s4" target="_blank">methods</a>), but no such effect was detected.</p

Group-wise Spearman rank correlations with CD25^bright/CD4⁺ frequencies.

<p>Group-wise Spearman rank correlations with CD25^bright/CD4⁺ frequencies.</p

<i>IL2RA</i> model properties.

<p><i>IL2RA</i> model properties.</p