Mediator of DNA Damage Checkpoint 1 (MDC1)

pTER_MDC1 (422-2)

High resolution pdf: pTER_MDC1 (422-2).pdf

Sequence file: pTER_MDC1 (422-2).seq

Addgene#:22191

Sequence verification file: 422-2BGHrev.ab1

Recommended E.coli strain for propagation: TOP10F'

Selection: Ampicillin and or Zeocin.

NOTE: This is NOT a Gateway vector and it is not compatible with any Destination vectors. It needs to be transfected and selected with Zeocin.

 

pENTR/pTER shMDC1 (441-1)

High-resolution pdf: pENTR/pTER shMDC1 (441-1).pdf

Sequence file: pENTR/pTER shDMC1 (441-1).seq

Addgene#: 22192

Sequence verification file: 441-1ENTRrev.ab1

Recommended E.coli strain for propagation: TOP1-F'.

Selection: Kanamycin.

Recommended Destination vectors: pLenti X1 series, pLenti X2 series, pLenti CMV GFP, pQCXI X2 series.

 

pLenti X1 Zeo/pTER shMDC1 (673-1)

High-resolution pdf: pLenti X1 Zeo/pTER shMDC1 (673-1).pdf

Sequence file: pLenti X1 Zeo/pTER shMDC1 (673-1).seq

Addgene #:22193

Recommended E.coli strain for propagation: Stbl3.

Selection: Zeocin (50 ug/ml).

 

pLenti X2 Neo/pTER shMDC1 (w24-2)

High-resolution pdf: pLenti X2 Neo/pTER shMDC1 (w24-2).pdf

Sequence file: pLenti X2 Neo/pTER shMDC1 (w24-2).seq

Addgene #:22195

Recommended E.coli strain for propagation: Stbl3.

Selection: Ampicillin.

 

pLenti X2 Zeo/pTER shMDC1 (764-1)

High-resolution pdf: pLenti X2 Zeo/pTER shMDC1 (764-1).pdf

Sequence file: pLentiX2 Zeo/pTER shMDC1 (764-1).seq

Addgene #: 22194

Recommended E.coli strain for propagation: Stbl3.

Selection: Zeocin (50 ug/ml).

 

MDC1 in pENTR4 (330-5)

High-resolution pdf: pENTR-MDC1 (330-5).pdf

Sequence file: pENTR-MDC1 (330-5).seq

Sequence verification files: 330-5ENTRforw.ab1, 330-5ENTRrev.ab1

Addgene #: 26427

Recommended E.coli strain for propagation: TOP10F'.

Selection:Kanamycin.

Compatible Destination vectors: pLenti CMV, CMV/TO, PGK, pQCX series.

pENTR4-GFP-MDC1 (770-7)

High-resolution pdf: pENTR4-GFP-MDC1 (770-7).pdf

Sequence file: pENTR4-GFP-MDC1 (770-7).seq

Addgene #: 26284

Recommended E.coli strain for propagation: TOP10F'.

Selection:Kanamycin.

Compatible Destination vectors: pLenti CMV, CMV/TO, PGK, pQCX series.

 

pLenti CMV/TO GFP-MDC1 (779-2)

High-resolution pdf: pLenti CMV/TO GFP-MDC1 (779-2).pdf

Sequence file: pLenti CMV/TO GFP-MDC1 (779-2).seq

Addgene #: 26285

Recommended E.coli strain for propagation: Sbtl3.

Selection: Ampicillin.

 

MCF10A cells were transduced with a lentivirus expressing GFP-MDC1 (pLenti CMV/TO GFP-MDC1). Cells were splitted and 10 000 cells per well were seeded on an eight-well immunofluorescence slide. The slide was irradiated with X-Rays (5 Gy) and incubated for one hour at 37oC. The cells were then fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 in PBS. The slide was blocked and incubated in the presence of an antibody against the phosphorylated form of H2AX (Gamma-H2AX, clone JBW301, Upstate). An anti-mouse Cy5 secondary antibody (Jackson immunochemicals) was used to detect the gamma-H2AX by immunofluorescence. The endogenous fluorescence of GFP was detected. As reported in the literature, MDC1 co-localizes with gamma-H2AX at sites of DNA damage detected by foci. GFP-MDC1 still localizes at foci in the absence of gamma-H2AX antibody (negative control for cross-reactivity and "channel bleeding"). PDF version of the slide can be downloaded here.