Id helix-loop-helix proteins in cell growth and differentiation
Başlık:
Id helix-loop-helix proteins in cell growth and differentiation
Yazar:
Craggs, Graham, author.
ISBN:
9780438043169
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (248 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 76-08C.
Özet:
Id proteins (Idl-4) represent a distinct class of helix-loop-helix (HLH) proteins that have been recognised as playing important roles in cell growth and differentiation. At the general level, Id proteins serve to repress differentiation-linked gene expression and promote cell cycle progression by antagonising basic helix-loop-helix (bHLH) transcription factors and members of the pRb tumour suppressor protein family. The roles of Id proteins in cell growth and differentiation have been extensively studied in vitro using cell line and biochemical models. In order to establish Id function in vivo, a collaborative project was initiated to inactivate all four Id genes in mice by gene targeting. This thesis describes the cloning and inactivation of the mouse Id1 and M3 genes. Genomic clones harbouring the murine Id1 and Id3 gene loci were isolated and the structure and sequence of each gene determined. Id3 was shown to consist of three exons and be closely related to the Id2 and Id4 genes. Idl1displayed a distinct organisation and is reasoned to be the ancestral /c/-gene. Transcriptional control elements were recognised in both gene loci. Of particular importance is an intronic E2F binding site located in the Id3 locus which is shared with the human homologue and might direct the cell cycle regulated expression of Id3. Gene tai-geting vectors for Id1 and Id3 were constructed which underwent homologous recombination with their respective loci in embryonic stem (ES) cells. A sensitive and robust PCR-based system was used to detect gene targeted ES cells in combination with Southern analysis. The Id3 locus was shown to be a hotspot for targeted recombination, with 20% of drug resistant ES cell clones harbouring an inactivated allele. Homologous recombination of the Id1 gene was less efficient with less than 1% of drug resistant ES cell clones containing a correctly targeted allele. The Id1+/- and IdS+/- ES cell clones were used to generate chimeric mice that were up to 85% ES cell-derived. However, germ line transmission of the ES genotype was not attained. Analysis of targeted and non-targeted ES cells revealed that chromosomal aberrations accumulated during their in vitro culture that was coincident with the loss of germ line colonisation. Published observations of Id1 and Id3 knockout mice revealed that functional complementation alleviated phenotypic abnormalities. However, double knockout mice were not viable and underwent embryonic death. In order to circumvent the lethal phenotype of double knockout mice, a project was established to conditionally inactivate the Id3 gene using the CRE-LOXP system. A targeting vector was constructed that will enable conditional targeting of the Id3 gene to be achieved. In a separate study, retroviral vectors were used to overexpress Id proteins in myeloid progenitor cells. The green fluorescent protein marker was shown to be a convenient marker for monitoring transduced cell populations. Id1 and Id3 proteins were shown to inhibit the formation of progenitor-derived myeloid colonies from mouse bone marrow. Furthermore, Id2 alone was shown to inhibit the survival of FDCP-mix myeloid cells. In conclusion, the constitutive inactivation and/or overexpression of Id genes is unlikely to uncover their in vivo role due to functional complementation within the gene family and promotion of apoptosis.
Notlar:
School code: 1543
Tüzel Kişi Ek Girişi:
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Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
---|---|---|---|
XX(684253.1) | 684253-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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