Transgenic Mice Research Based On BAC Harbouring The Mutant Human α-Globin Gene Cluster

The human globin gene family consists ofα- andβ-globin gene clusters. Theβ-globin gene cluster is on the short arm of chromosome 11 and in the order of 5'-ε-Gγ-Aγ-Ψβ-δ-β-3'. Theα-globin gene cluster is located near the telomeric region of chromosome 16 and its members are arrayed in the order of developmental switching: 5'-ζ-Ψζ-Ψα₂-Ψα₁.α₂-α₁-θ-3'. Theζ-globin gene is expressed at the embryonic stage, whereas the twoα-globin genes,α₂ andα₁, are turned on throughout the fetal and adult life. Theθ-globin gene is also adult stage-specific but its level of expression is very low and its function is still unknown. The expressions of globin genes are characterized by 1) sequential expression of individual gene in each cluster; 2) erythroid-specific and developmental stage-specific expression; 3) balance betweenα-like andβ-like globin chains.The expressions of globin gene clusters are regulated on levels of epigenetics, transcription, posttranscription, translation, and so on. The transcription regulation of globin genes is mainly related to local and distal cis-acting elements and cellular trans-acting factors. There are five DNase I hypersensitive sites upstream of the human a-globin gene cluster, designated HS-4, HS-8, HS-10, HS-33 and HS-40 according to their distance from theζmRNA cap site. In previous studies, HS-40 has been shown to confer high levels of erythroid-specific expression of linked humanζ- orα-globin genes in transgenic mice or stably integrated cell lines. Though HS-40 has been shown to act as an enhancer-like element, the other four HS sites have no effect on the expression of human a-globin genes both in mouse erythroleukemia (MEL) cells and transgenic mice. However, the deletion of mHS-26, whose structure and function are thought to be similar with human HS-40, has led to an unexpectedly mildα-thalassemia phenotype; and there is no evidence thatα-thalassemia results from the natural deletion of the single HS-40. So the functional roles and mode of these elements should be further defined on the whole cluster level.Studies of expression regulation of eukaryotic genes in transgenic mice permit not only the devices of experiments on molecular level but also the observation of the involvement of almost all the factors affecting gene expression and investigations of in vivo effect of gene regulations. Transgenic technique can be applied to set up transgenic models for human genetic diseases from which expression regulation of abnormal genes are demonstrated and many pre-clinical data for human gene therapy will be provided. Bacterial artificial chromosome (BAC) vector has been applied in large fragment transgenic research of gene cluster. Based on the Escherichia Coli F factor, BAC has high stability and minimal chimerism, it can propagate up to 300kb foreign DNA, which is large enough to cover many gene clusters. BAC can be used to perform research work with ease and reproducibility, even for gene cluster containing high percentage of repeat sequences like humanα-globin gene cluster. Mutation analysis of BAC DNA will allow us to study the relationship between the structure and function of the entire gene cluster and may reveal other yet unknown regulatory elements that control transcription.Previously, one 117kb BAC clone (191K2) containing the entire humanα-globin gene locus has been obtained, and HS-40 deletion mutant BAC (αMI) was also gained.In this study, to define the functional roles and mode of the upstream regulatory elements ofα-globin gene cluster,αMâ…¡was obtained, in which the 31.7kb sequence including HS-33, HS-10,HS-8 and HS-4 was deleted by defective prophage mediated by the homologous recombination;αMâ…¢was also constructed, in which the 35.4kb sequence including HS-40,HS-33,HS-10,HS-8 and HS-4 was deleted by the method of temperature sensitive shuttle vector mediated homologous recombination.After preparation and purification,αMâ… ,αMâ…¡andαMâ…¢BAC DNA were linearized with Not I digestion, and the insert DNA fragments were separated from the pBeloBAC11 vector by Sepharose CL-4B filter. Then, RNase Protection Assays were performed to detect the expression ofα-like globin genes in different developmental stages and tissues. The results show that: 1) 6 transgenic mice harboring completeαM I BAC DNA were got. After HS-40 deletion, hζ- and hα-globin genes expressed on low levels, and the per copy expression levels were much lower than the normalα-BAC transgenic mice's. The expression was copy number independent and integration-site dependent in the transgenic lines, and tissue- and developmental-stage specific expression mode was normal; 2) 5 transgenic mice harboring completeαMâ…¡BAC DNA were got. The deletion of HS-4-33 had different effect on hζ- and hα-globin genes: the expression level of hζ-globin gene was comparable with that of normalα-BAC transgenic mice, while hα-globin gene expression decreased. The expression was still copy number independent and integration-site dependent in the transgenic lines, and tissue- and developmental-stage specific mode was not affected; 3) 7 transgenic mice harboring completeαMâ…¡BAC DNA were got. Both in different erythroid and nonerythroid tissues, hα-like globin genes were not expressed at different development stages. 4) the expression of ha-like globin genes in transgenic mice did not disturb mouse endogenousα-like globin genes expression.Conclusion: 1) The correct mutant could be obtained as long as the homologous recombination method is suitable, even to the humanα-globin gene cluster containing highly repetitive sequences; 2) the expression of humanα-like globin genes in transgenic mice did not disturb mouse endogenous expression, so humanα-like globin genes expression levels could be correctly defined by mouse endogenousα-like globin genes expression at the corresponding stages; 3) the tissue- and development- specific expression of humanα-globin gene cluster was correct even HS-40 or HS-4～33 was deleted, thus at least the humanα-globin gene cluster expression mode does not depend on some single upstream regulatory element; 4) the deletion of HS-4～40 resulted in the silence of humanα-like globin genes, and it implies that this sequence is pivotal for humanα-like globin genes expression on transcriptional level; 5) humanα-like globin genes expression level decreased after the deletion of HS-40 core sequence, and though HS-40 has important roles on humanα-like globin genes transcriptional expression, it should not be the only element who keeps the humanα-like globin genes expression; 6) after HS-4～33 was deleted, the expression of humanα-globin gene decreased, however humanζ-gliobin gene still kept high transcriptional level. This illuminates that some element(s) in this sequence could affect the transcriptional expression level of humanα-globin gene expression, whereas has no effect on humanζ-gliobin gene, that is this sequence has the development-specific regulation role.All together our results indicate that, both HS-4～33 and HS-40 are significant for the humanα-like globin genes, but whichever is not enough to regulate the humanα- globin gene cluster by itself. So we speculate that : similar to theβ-LCR looping model, HS-4～40(upstream regulatory elements of humanα-globin gene cluster), also as a loop, act with the promoters of downstream structural genes to regulate the transcriptional expression of the humanα-like globin genes. And that HS-40 and HS-4～33 have different roles on the regulation: HS-40 has important enhancement on humanα-like globin genes at different stages; HS-4～33 show the development-specific regulation. It only enhances the fetal/adult stage humanα-globin gene, and it will rescue the expression of embryonic stage humanζ-globin gene only ifHS-40 is deleted.