Method for Regulating Cell Proliferation
a cell proliferation and cell technology, applied in the field of cell proliferation regulation, can solve the problems of large amount of defective proteins produced, disrupted function of the er,
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example 1
Establishment of BBF2H7 Knockout Mouse
(1) Preparation of a Targeting Vector
[0155]A PCR (35 cycles of denature: 94° C. for 1 minute, annealing: 55° C. for 1 minute, and extension: 72° C. for 3 minutes) was performed with the genomic DNA from 129 mouse as a template and primers prepared based on the genomic sequence of the mouse BBF2H7 (ENSMUSG00000038648). DNA fragments of 1.5 kb (short arm) and 6 kb (long arm) around exon 2 of the BBF2H7 gene were isolated. The following primers were used:
for the short arm,(SEQ ID NO.: 5)5′-GCGGCCGCTTCGACACTTTGTCTGCCACTC-3′and(SEQ ID NO.: 6)5′-CTCGAGTCACTCCGAGAAGTGCTGCAAGAAGC-3′;for the long arm,(SEQ ID NO.: 7)5′-CAGAGATGCCCTGAGATCAGCTG-3′and(SEQ ID NO.: 8)5′-GGTACCCTACACCATGCGCCACCAGCCATG-3′.
[0156]The short and long arm DNA fragments were sequenced to confirm no nucleotide substitution was occurred.
[0157]Subsequently, the DNA fragments were inserted into the NotI-XhoI site (short arm) and the KpnI-KpnI site (long arm) of pPNT1.1 (Cell 1991 June 28,...
example 2
Proliferation of Primary Cultured Chondrocytes from the BBF2H7 Knockout Mouse
[0163]Primary cultured chondrocytes were derived from the wild-type (WT) mouse (C57BL / 6CR SLC mouse) and the Bbf2h7-deficient (Bbf2h7− / −) mouse (See Saito et al., Nat. Cell Biol. 2009, 11:1197-1204.). Proliferation of the cells was investigated with BrdU-incorporation assay. The assay revealed that BrdU-incorporation was significantly reduced in the Bbf2h7-deficient cells. This result indicates that the cell growth rate was reduced in the Bdf2h7-deficient cells (FIG. 5).
example 3
Generation of Anti-BBF2H7 Antibody
[0164]In order to generate an anti-human BB2H7 C terminus antibody, a mouse was immunized with a partial peptide (IYEEHSPPEESSSPGSAGELGGWDRGSSLLRVSGLESRPDVDLPHFIISNETSLEKSV LLE (SEQ ID NO.: 12)) as an antigen. Spleen cells were removed from the immunized mouse and fused to myeloma cells. The fused cells were screened for cells producing an antibody which binds to the antigen.
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