A humanized mouse model for a common beta0-thalassemia mutation

Accurate animal models that recapitulate the phenotype and genotype of patients with beta-thalassemia would enable the development of a range of possible therapeutic approaches. Here we report the generation of a mouse model carrying the codons 41-42 (-TTCT) beta-thalassemia mutation in the intact human beta-globin locus. This mutation accounts for approximately 40% of beta-thalassemia mutations in southern China and Thailand. We demonstrate a low level of production of gamma-globins from the mutant locus in day 18 embryos, as well as production of mutant human beta-globin mRNA. However, in contrast to transgenic mice carrying the normal human beta-globin locus, 4-bp deletion mice fail to show any phenotypic complementation of the knockout mutation of both murine beta-globin genes. Our studies suggest that this is a valuable model for gene correction in hemopoietic stem cells and for studying the effects of HbF inducers in vivo in a "humanized" thalassemic environment.     

Insertion of common mutations into the human beta-globin locus using GET Recombination and an EcoRI endonuclease counterselection cassette

A large number of mutations have been described in the human beta-globin locus causing thalassemia or various hemoglobinopathies. However, only a very limited number of these mutations have been studied in animal model systems in the context of the human beta-globin locus. We report here the use of the GET Recombination system with an EcoRI/Kan(R) counterselection cassette to facilitate the introduction of the HbE (codon 26, GAG-->AAG mutation and the codon 41-42 (-TTCT) deletion, two mutations found in high frequency in South-East Asia, into the human beta-globin locus. The counterselection cassette was first inserted into the target sequence in the beta-globin gene, and then a PCR fragment carrying the required modification was used to replace it. Efficient counterselection depends upon the tight regulation of the highly toxic EcoRI endonuclease gene by expression of lacI(q). Induction by IPTG during counterselection efficiently eliminates non-recombinant bacterial clones. The technique can be performed on any known gene sequence using current BAC technology, allowing identification and comparative functional analysis of key regulatory elements, and the development of accurate animal models for human genetic disorders.     

Transgene copy number-dependent rescue of murine beta-globin knockout mice carrying a 183 kb human beta-globin BAC genomic fragment

We report the generation and characterisation of the first transgenic mice exclusively expressing normal human beta-globin ((hu)beta-globin) from a 183 kb genomic fragment. Four independent lines were generated, each containing 2-6 copies of the (hu)beta-globin locus at a single integration site. Steady state levels of (hu)beta-globin protein were dependent on transgene copy number, but independent of the site of integration. Hemizygosity for the transgene on a heterozygous knockout background ((hu)beta(+/0), (mu)beta(th-3/+)) complemented fully the hematological abnormalities associated with the heterozygous knockout mutation in all four lines. Importantly, the rescue of the embryonic lethal phenotype that is characteristic of homozygosity for the knockout mutation was also demonstrated in two transgenic lines that were homozygous for two copies of the (hu)beta-globin locus, and in one transgenic line, which was hemizygous for six copies of the (hu)beta-globin locus. Our results illustrate the importance of transgene copy number determination and of the hemizygosity/homozygosity status in phenotypic complementation studies of transgenic mice containing large heterologous transgenes. Transgenic mouse colonies with 100% (hu)beta-globin production from the intact (hu)beta-globin locus have been established and will be invaluable in comparative and gene therapy studies with mouse models containing specific beta-thalassemia mutations in the (hu)beta-globin locus.     

A humanized BAC transgenic/knockout mouse model for HbE/beta-thalassemia

Hemoglobin E (HbE) is caused by a G-->A mutation at codon 26 of the beta-globin gene, which substitutes Glu-->Lys. This mutation gives rise to functional but unstable hemoglobin and activates a cryptic splice site causing mild anemia. HbE reaches a carrier frequency of 60-80% in some Southeast Asian populations. HbE causes serious disease when co-inherited with a beta-thalassemia mutation. In this study, we report the creation and evaluation of humanized transgenic mice containing the beta(E) mutation in the context of the human beta-globin locus. Developmental expression of the human beta(E) locus transgene partially complements the hematological abnormalities in heterozygous knockout mice ((mu)beta(th-3/+)) and rescues the embryonic lethality of homozygous knockout mice ((mu)beta(th-3/th-3)). The phenotype of rescued mice was dependent on the transgene copy number. This mouse model displays hematological abnormalities similar to HbE/beta-thalassemia patients and represent an ideal in vivo model system for pathophysiological studies and evaluation of novel therapies.     

Factors affecting immobilization of heavy metals by purple nonsulfur bacteria isolated from contaminated shrimp ponds

In order to remove heavy metals (HMs) from contaminated shrimp pond at the highest concentrations found of; 0.75 mg/l Cd²⁺, 62.63 mg/l Pb²⁺, 34.60 mg/l Cu²⁺ and 58.50 mg/l Zn²⁺, two strains of purple nonsulfur bacteria isolated from shrimp ponds (NW16 and KMS24) were investigated for their ability to immobilize HMs in 3% NaCl in both microaerobic-light and aerobic-dark conditions. Based on metabolic inhibition and metabolic-dependent studies, it was concluded that both strains removed HMs using biosorption and also bioaccumulation. The efficiency of removal by both strains with both incubating conditions tested was in the order of lead (Pb) > copper (Cu) > zinc (Zn) > cadmium (Cd). Optimal conditions for removal of HMs by strain NW16 were; cells in the log phase at 4.5 mg DCW/ml, pH 6.0, and 30°C for 30 min. With microaerobic-light conditions, the relative percent removal of HMs was: Pb, 83; Cu, 59; Zn, 39; Cd, 23 and slightly more with the aerobic-dark conditions (Pb, 90; Cu, 69; Zn, 46; Cd, 28). Cells in the log phase at 5.0 mg DCW/ml, pH 5.5, and 35°C for 45 min were optimal conditions for strain KMS24 and there were no significant differences for the removal percentages of HMs with either incubating conditions (averages: Pb, 96; Cu, 75; Zn, 46; Cd, 30). The presence of Ca²⁺ and Mg²⁺ significantly decreased the removal capacity of HMs for both strains.