Expression of tight junction protein ZO-2 in mouse preimplantation embryos
Expression of tight junction protein ZO-2 in mouse preimplantation embryos
RT-PCR analyses detected ZO-2 mRNA in three single mouse late blastocysts. The resultant cDNA fragment was cloned and sequenced revealing 98% homology with available mouse cDNA sequences. This partial mouse cDNA clone was used to produce fusion protein for the generation of polyclonal rabbit-anti-mouse ZO-2 antibodies. Polyclonal antibodies were also raised against a ZO-2-specific synthetic peptide. Anti-fusion protein antibodies detected a prominent band at �160kDa which was not present in respective blots probed with preimmune serum (PI). Wholeamount staining of mouse preimplantation embryos reveal characteristic zonular staining at cell contacts indicative of a TJ-like staining pattern not seen in embryos stained with the respective PI serum. However, the results gained from using the polyclonal anti-mouse ZO-2 antibodies were highly variable.
ZO-2 expression was studied in mouse preimplantation embryos, post-implantation embryos, Madin Darby canine kidney cells (MDCK), and in cryostat sections of mature mouse tissues using a commercially available anti-human ZO-2 antibody. ZO-2 protein was detected in TJ at cell contact sites in MDCK cells, and in the TJ or oral epithelial cells, dermis, and intestine in mouse postimplantation embryos, and in the TJ of adult seminiferous epithelium and bile caniculi of the liver. ZO-2 expression was first detected in cumulus cells and unfertilised eggs through Western blotting whereas the first evidence of membrane-associated ZO-2 staining was seen at the 16-cell stage. Cell couplet experiments showed that membrane assembly of ZO-2 occurs first at the late 16-cell stage/early 32-cell stage. Data suggested that ZO-2 colocalises with E-cadherin at the 16-cell stage and shifts to colocalise with ZO-1 and occludin at the late morulae stage.
University of Southampton
1999
Nowak, Rachael Louise
(1999)
Expression of tight junction protein ZO-2 in mouse preimplantation embryos.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
RT-PCR analyses detected ZO-2 mRNA in three single mouse late blastocysts. The resultant cDNA fragment was cloned and sequenced revealing 98% homology with available mouse cDNA sequences. This partial mouse cDNA clone was used to produce fusion protein for the generation of polyclonal rabbit-anti-mouse ZO-2 antibodies. Polyclonal antibodies were also raised against a ZO-2-specific synthetic peptide. Anti-fusion protein antibodies detected a prominent band at �160kDa which was not present in respective blots probed with preimmune serum (PI). Wholeamount staining of mouse preimplantation embryos reveal characteristic zonular staining at cell contacts indicative of a TJ-like staining pattern not seen in embryos stained with the respective PI serum. However, the results gained from using the polyclonal anti-mouse ZO-2 antibodies were highly variable.
ZO-2 expression was studied in mouse preimplantation embryos, post-implantation embryos, Madin Darby canine kidney cells (MDCK), and in cryostat sections of mature mouse tissues using a commercially available anti-human ZO-2 antibody. ZO-2 protein was detected in TJ at cell contact sites in MDCK cells, and in the TJ or oral epithelial cells, dermis, and intestine in mouse postimplantation embryos, and in the TJ of adult seminiferous epithelium and bile caniculi of the liver. ZO-2 expression was first detected in cumulus cells and unfertilised eggs through Western blotting whereas the first evidence of membrane-associated ZO-2 staining was seen at the 16-cell stage. Cell couplet experiments showed that membrane assembly of ZO-2 occurs first at the late 16-cell stage/early 32-cell stage. Data suggested that ZO-2 colocalises with E-cadherin at the 16-cell stage and shifts to colocalise with ZO-1 and occludin at the late morulae stage.
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Published date: 1999
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Local EPrints ID: 464054
URI: http://eprints.soton.ac.uk/id/eprint/464054
PURE UUID: 6c358d45-f426-4c07-a636-e3691b9e99c0
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Date deposited: 04 Jul 2022 21:01
Last modified: 04 Jul 2022 21:01
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Author:
Rachael Louise Nowak
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