Created to destroy all three 59 AUG-initiation products of X while not affecting the cap coding sequence, used to understand the role of the X gene in AAV2 DNA replication.
3
X-positive-293 cell lines (293-X-B, 293-X-K)
Generated by transfecting 293 cells with pCI/X/Neo and selecting with G418, used to study the contribution of the X gene to AAV2 DNA replication.
4
Ad5 helper plasmid (pHelper)
Used to supply necessary Adenovirus helper functions in 293 cells during AAV2 replication assays.
5
Anti-98-X polyclonal antibody
Used to detect the X protein in a Western blot from Ad5-infected 293 cells.
6
pCI/X/Neo
Used to transfect 293 cells to establish cell lines expressing the X gene, facilitating studies on the X gene's effect on AAV2 replication.
7
Primary human foreskin keratinocytes (PHFK)
Used in organotypic epithelial raft cultures to investigate autonomous AAV2 replication in differentiating skin cells.
8
G418
Used for selecting 293-X-Neo resistant cell lines after transfection with pCI/X/Neo.
9
Fugene6
Used for transfecting primary human foreskin keratinocytes and 293 cells with AAV and X gene plasmids.
10
32P-labeled DNA probe
Used for Southern blot to detect AAV2 DNA replication in various experiments.
11
Trizol reagent
Used for isolating total RNA from epithelial organotypic rafts for RT-PCR analysis.
12
Oligotex mRNA Mini Kit
Used to isolate mRNA from total RNA samples as a preliminary step for RT-PCR.
13
Rabbit polyclonal antibodies against a peptide derived from aa 98–111 of AAV2 X
Used to probe Western blots for identification of the X protein in protein extracts from 293 cells.
14
Primary human foreskin keratinocytes transfected with AAV/X/Neo or AAV/Neo plasmids
Used in the skin raft culture system to analyze the effects of X on AAV2 autonomous DNA replication.
15
Total DNA isolated using lysis buffer with proteinase K
Used to phenol extract and ethanol precipitate DNA from raft tissues for Southern blot analysis.
16
Ad5 (Adenovirus type 5)
Used to provide helper virus functions in 293 cells during AAV2 DNA replication assays.
17
pAAV-RC
Used as a control in comparative assays against pAAV-RC-3Xneg to analyze AAV2 DNA replication and virion production.
18
AAV2 viral stocks
Infected to PHFK in the absence of adeno-, herpes-, or papilloma-virus helper virus to study X gene function in the natural host tissue.
19
E medium
Used to maintain epithelial organotypic rafts (skin raft cultures) during stratification and differentiation.
20
pHelper, pAAV-RC, pAAV-RC-3Xneg, pAAV/eGFP
Used in the standard 293 cell triple transfection procedure to generate rAAV/eGFP virus.
21
DNase I
Used in virion DNA analysis for digesting non-encapsidated DNA before extracting virion DNA.
22
Proteinase K
Used for digesting proteins in various DNA extraction protocols during the study.
23
Pronase K
Used to digest proteins in total cellular DNA extraction for Southern blot analysis.
