Functions Of Defined Transcription Factors In Mammalian Growth, Development And Cellular Reprogramming

In this study, PCR-SSCP, PCR-RFLP, DNA sequencing, protein expression and purification, cell transfection and in vitro transcription were applied to verify the functions of specific transcription factor in mammalian growth, development and the process of somatic cell reprogramming. At the meantime, 3 different levels (gene, protein and RNA) of specific induced factors cocktail were utilized in this dissertation. The main results were summarized as followings:1. The genetic variation of two Pituitary transcription factor genes bPOU1F1 and bPROP1 and their associations with economic traits in bovine.â‘´Three novel single nucleotide polymorphisms (SNPs) in exon 2 and one novel missense (NM₁74579:c.1201C>T) mutation in exon 6 at the POU1F1 locus in 963 Chinese cattle belonging to eight breeds were reported. The missense results in p.S284F, namely, Ser (TCT) > Phe (TTT) at position 284 of the mature protein of POU1F1. No significant associations of these genetic variations with body weight and average daily gain for different growth periods (6, 12, 18, and 24 months old) were observed (P>0.05), as well as for body sizes (P>0.05).⑵Five novel single nucleotide polymorphisms (SNPs) were found in 5'flanking region, exon 1 and intron 1 of the prophet of PIT1 (PROP1) gene in 606 unrelated cattle which belonged to five cattle breeds in China.2. Human iPS cells were reprogrammed from somatic cells by lentiviral expressed transcription factors using SNL fibroblasts as feeders. These iPS cells expressed common pluripotency markers, displayed human embryonic stem cells (hES) morphology and unmethylated promoters of Nanog and Oct4. These data demonstrate that SNL feeder cells can support the derivation and maintenance of human iPS cells.3. For the purpose of human therapeutic applications, the integration of viral transgenes into the genome is unlikely to be accepted.â‘´Recombinant transcription factor proteins in E. coli (Oct4, Sox2, c-Myc and Klf4) carrying the cell penetrating TAT domain from HIV1 were produced. The purified proteins were able to enter into mammalian cells when added to tissue culture medium but appeared not to translocate to the nucleus. Further investigation indicated that most of the protein was tied up in the endosomes and was unavailable for reprogramming.⑵In vitro transcribed induced factors (Oct4,Sox2 and SV40 T) mRNA were utilized to reprogram fibroblasts. 5'UTR and 3'UTR of Globin gene were used for stabilization in vitro transcribed mRNA following in vitro capping and poly(A) tailing. Target factors'expressions can be detected in 293 cells and IMR90 cells after mRNA transfection and all the expressed protein were localized in the nucleus. Endogenous Nanog expression was inducted by mRNA cock-tail transfection.