Evaluation of CRE-mediated excision approaches in Arabidopsis thaliana

The ability of the CRE recombinase to catalyze excision of a DNA fragment flanked by directly repeated lox sites has been exploited to modify gene expression and proved to function well in particular case studies. However, very often variability in CRE expression and differences in efficiency of CRE-mediated recombination are observed. Here, various approaches were investigated to reproducibly obtain optimal CRE activity. CRE recombination was analyzed either by transforming the CRE T-DNA into plants containing a lox-flanked fragment or by transforming a T-DNA harboring a lox-flanked fragment into plants producing the CRE recombinase. Although somatic CRE-mediated excision of a lox-flanked fragment was obtained in all transformants, a variable amount of germline-transmitted deletions was found among different independent transformants, irrespective of the orientation of transformation. Also, the efficiency of CRE-mediated excision correlated well with the CRE mRNA level. In addition, CRE-mediated fragment excision was compared after floral dip and after root tissue transformation when transforming in a CRE-expressing background. Importantly, less CRE activity was needed to excise the lox-flanked fragment from the transferred T-DNA after root tissue transformation than after floral dip transformation. We hypothesize that this is correlated with the lower T-DNA copy number inserted during root transformation as compared to floral dip transformation

Generation of Single-Copy T-DNA Transformants in Arabidopsis by the CRE/loxP Recombination-Mediated Resolution System1

We investigated whether complex T-DNA loci, often resulting in low transgene expression, can be resolved efficiently into single copies by CRE/loxP-mediated recombination. An SB-loxP T-DNA, containing two invertedly oriented loxP sequences located inside and immediately adjacent to the T-DNA border ends, was constructed. Regardless of the orientation and number of SB-loxP-derived T-DNAs integrated at one locus, recombination between the outermost loxP sequences in direct orientation should resolve multiple copies into a single T-DNA copy. Seven transformants with a complex SB-loxP locus were crossed with a CRE-expressing plant. In three hybrids, the complex T-DNA locus was reduced efficiently to a single-copy locus. Upon segregation of the CRE recombinase gene, only the simplified T-DNA locus was found in the progeny, demonstrating DNA had been excised efficiently in the progenitor cells of the gametes. In the two transformants with an inverted T-DNA repeat, the T-DNA resolution was accompanied by at least a 10-fold enhanced transgene expression. Therefore, the resolution of complex loci to a single-copy T-DNA insert by the CRE/loxP recombination system can become a valuable method for the production of elite transgenic Arabidopsis thaliana plants that are less prone to gene silencing

IL12RB2 Gene is Associated with the age of Type 1 Diabetes Onset in Croatian Family Trios

BACKGROUND: Common complex diseases are influenced by both genetic and environmental factors. Many genetic factors overlap between various autoimmune diseases. The aim of the present study is to determine whether four genetic variants known to be risk variants for several autoimmune diseases could be associated with an increased susceptibility to type 1 diabetes mellitus. METHODS AND FINDINGS: We genotyped four genetic variants (rs2358817, rs1049550, rs6679356, rs9865818) within VTCN1, ANXA11, IL12RB2 and LPP genes respectively, in 265 T1DM family trios in Croatian population. We did not detect association of these polymorphisms with T1DM. However, quantitative transmission disequilibrium test (QTDT, orthogonal model) revealed a significant association between the age of onset of T1DM and IL12RB2 rs6679356 variant. An earlier onset of T1DM was associated with the rs6679356 minor dominant allele C (p = 0.005). The association remained significant even after the Bonferroni correction for multiple testing and permutation. CONCLUSIONS: Variants originally associated with juvenile idiopathic arthritis (VTCN1 gene), sarcoidosis (ANXA11 gene), primary biliary cirrhosis (IL12RB2 gene) and celiac disease (LPP gene) were not associated with type 1 diabetes in our dataset. Nevertheless, association of IL12RB2 rs6679356 polymorphism with the age of T1DM onset suggests that this gene plays a role in defining the time of disease onset

Incidence of Type 1 Diabetes Mellitus in 0 to 14-yr-old Children in Croatia – 2004 to 2012 Study

BACKGROUND: The incidence of type 1 diabetes mellitus (T1DM) among children and adolescents increased during the last 50 yr. The T1DM incidence in Croatia was 8.87/100.000/yr over 1995-2003, with an annual increase of 9%, which placed Croatia among countries with moderate risk for T1DM. AIM: To investigate incidence rates and trends of T1DM from 2004 to 2012 in 0 to 14-yr-old Croatian children, and to compare the results with previous studies in Croatia and other European countries. METHODS: T1DM crude incidence rates are estimated for the entire group and three subgroups: 0-4, 5-9, and 10-14 yr. Standardized incidence is calculated using the method of direct standardization according to World Health Organization (WHO) standard world population. The incidence rates by gender, age groups, seasonality, and calendar year, and their interactions were analyzed using Poisson regression model. RESULTS: A total of 1066 cases were ascertained over 2004-2012. The standardized incidence was 17.23/100.000/yr (95% CI: 16.19-18.26), with no significant differences in incidence rates or trends between boys and girls. Statistically significant annual increase of 5.87% (p < 0.001) was found for the whole group, and for the subgroups 5-9 yr (6.82%; p < 0.001) and 10-14 yr (7.47%; p < 0.001). In the youngest subgroup (0-4 yr), annual increase was lower (2.43%; p = 0338) and not statistically significant. CONCLUSION: The incidence of childhood T1DM is increasing in Croatia, thus placing Croatia among countries with high risk for T1DM. The annual increment of 5.87% is considerably lower than 9.0% reported earlier, but still higher than the European average (3.9%). The increase in incidence ceased in youngest children

Quantitative transmission disequilibrium analysis (Abecasis’s orthogonal test) in 262 family trios with age of T1DM onset as a quantitative variable.

*<p>Nominal p values are shown uncorrected for multiple testing. The Bonferroni-corrected significance threshold was p = 0.0125.</p>**<p>Empirical p-values were obtained using 10000 Monte Carlo permutations. Empirical significance threshold of 0.05 corresponded to a p-value of <0.0128.</p

Transmission disequilibrium analysis for <i>IL12RB, ANXA11, VTCN1</i> and <i>LPP</i> gene SNPs in 265 family trios (T:U – copies of the minor allele transmitted (T) and untransmitted (U) from heterozygous parents to affected offspring<i>).</i>

*<p>Nominal p values are shown uncorrected for multiple testing. The Bonferroni-corrected significance threshold is p = 0.0125.</p

Gene silencing induced by hairpin or inverted repeated sense transgenes varies among promoters and cell types

In transgenic calli and different tissues of Arabidopsis thaliana plants, the in trans silencing capacity of a 35S-beta-glucuronidase (GUS) hairpin RNA construct was investigated on a target GUS gene, under the control of the 35S, a WRKY or several cell cycle-specific promoters. GUS histochemical staining patterns were analyzed in all tissues of the parental lines and supertransformants harboring the hairpin construct. Quantitative GUS activity measurements determined GUS suppression by a 35S-GUS hairpin or inverted repeated GUS transgenes in leaves and calli. In some supertransformants, GUS-based staining disappeared in all tissues, including calli. In most supertransformants, however, a significant reduction was found in mature roots and leaves, but residual GUS activity was observed in the root tips, young leaves and calli. In leaves of most hairpin RNA supertransformants, the GUS activity was reduced by c. 1000-fold or more, but, in derived calli, generally by less than 200-fold. The silencing efficiency of inverted repeated sense transgenes was similar to that of a hairpin RNA construct in leaves, but weaker in calli. These results imply that the tissue type, nature of the silencing inducer locus and the differential expression of the targeted gene codetermine the silencing efficiency