Shared Resources and Protocols

Shared Resources

CIHD centralized resource site for sharing and accessing useful hematopoiesis related, or Iron and Heme metabolism relevant research tools and protocols

 

If you would like to request one of the items listed below, or if you have items that you would like to share with the CIHD community, please contact Hector Bergonia at hector.bergonia@hsc.utah.edu.

 

CIHD currently offers the following resources:

 

Lentiviral and transfection CRISPR control plasmids with puromyicin, blasticidin, GFP or mCherry selection.

Offered through the CIHD Mutation Generation and Detection Core:

 

 

 

Human Orfeome collection:  Over 8000 full-length human open reading frames (ORFs) from the Mammalian Genome Collection have been cloned into the Gateway Entry vector as described by Rual et al. (2004). These clones are now available free of charge to Health Sciences researchers as part of a partnership amongst the Huntsman Cancer Institute, HSC Core Research Facilities and individual contributing investigators.  For more information, including a list of available clones, please see http://cores.utah.edu/human-orf/

 

Plasmids to facilitate mitochondrial purification

David Sabatini kindly gave permission for these to be listed here

3XHA-EGFP-OMP25

3XMyc-EGFP-OMP25

 

Another way to separate mitochondrial from cytosolic fractions used by some of our researchers, is use of the ThermoScientific Mitochondrial Isolation Kit for Mammalian Cells.

 

Diane Ward has a large database of plasmids and strains that are related to iron metabolism, that she is willing to share.  For example:

cyc1-lacZ (JK0800)

HMG-lacZ (JK0583 from Jasper Rine)

AFT1up

FET3-lacZ

Mrs3-lacZ

Mrs4-lacZ

Ccc1-lacZ

cytosolic GDO-flag

ABCB10 constructs (mouse, zebrafish & human)

Mouse & human Mfrn1 & Mfrn2 constructs

Mouse & human Fpn-GFP

mouse Fpn-mcherry

DMT1-GFP

Dr. Ward also has many other yeast, iron-related genes in high and low copy vectors, and we would be happy to check if there is something else you are looking for.

 

Yeast Deletion Collections:

Shared through Diane Ward lab:

homozygous diploid deletion collection, S288c BY4743 yeast strains

heterozygous diploid deletion collection

 

Human Orfeome collection:  Over 8000 full-length human open reading frames (ORFs) from the Mammalian Genome Collection have been cloned into the Gateway Entry vector as described by Rual et al. (2004). These clones are now available free of charge to Health Sciences researchers as part of a partnership amongst the Huntsman Cancer Institute, HSC Core Research Facilities and individual contributing investigators.  For more information, including a list of available clones, and contact information for clone requests, please see http://cores.utah.edu/human-orf/

 

If you would like to request one of these items, or if you have items that you would like to share with the CIHD community, please contact Hector Bergonia at hector.bergonia@hsc.utah.ed

 

Please note, a simple MTA will be required for external CIHD members (outside of U of Utah) and in cases where the construct was generated outside of a CIHD lab, we ask that you contact the appropriate labs for permission.

 

We are interested in collecting and offering resources such as:

 

Cell lines (KO fibroblasts, CRISPR cell lines, floxed cell lines)

Plasmids

Antibodies

 

Thank you for your support!

Shared Protocols

Metabolomics Core Facility Protocols can be found here:

 

Iron and Heme Core Facility Protocols can be found here:

 

Protocols for heme quantitation:

Provided by Dennis Winge

 

β-Galactosidase Heme Reporter Assay– Cellular heme levels in whole cells may be quantified indirectly using heme responsive lacZ-reporter constructs. Hap1 is a heme-responsive transcriptional activator (Zhang and Guarente, 1995). Heme induced dimerization activates Hap1 for transcriptional activation of CYC1 (Zhang et al., 1993) (Thorsness et al., 1989). Heme is a negative regulator of HMG2 and Hap1 partially mediates this effect (Thorsness et al., 1989). CYC1/lacZ and HMG2/lacZ fusion genes should be separately transformed into a given yeast strain to assess heme levels. The heme-responsive β-galactosidase reporter plasmid under the control of the CYC1 promoter (pCYC1-lacZ fusion, obtained from Dr. L. Zhang). Cells transformed with pCYC1-lacZ plasmid are grown to early to mid-log phase in the appropriate SC auxotrophic medium supplemented with 2% raffinose. Then β-galactosidase is assayed according to published methods shown below:

 

Zhang, L., and Guarente, L. (1995) Heme binds to a short sequence that

serves a regulatory function in diverse proteins. EMBO J. 14, 313–320

 

Thorsness, M., Schafer, W., D’Ari, L., and Rine, J. (1989) Positive and

negative transcriptional control by heme of genes encoding 3-hydroxy-3-

methylglutaryl coenzyme A reductase in Saccharomyces cerevisiae. Mol.

Cell. Biol. 9, 5702–5712

 

Heme Quantitation– Total mitochondrial hemes b and PPIX may be analyzed using a modified version of the acid-acetone extraction of hemes and subsequent analysis using C18 reverse-phase HPLC. Briefly, 0.75 to 1 mg of purified mitochondria are pelleted as above and resuspended in 100 to 200 μL acetone/2.5% HCl solution. The volume of the solution is minimized to about 10 to 20 μL using a vacuum centrifuge and resuspended in 200 μL HPLC starting buffer A (25% acetonitrile + 0.1% TFA). Insoluble material is clarified using a second centrifugation and the supernatant is applied to a SunFire C18 column (4.6 mm Å~ 150 mm) and eluted at 1 mL/min using a 25-50% gradient for the first 4 mL and 50-75% for the 22 mL. Elution of heme and PPIX are monitored at 400 nm. For additional details see the reference below:

 

Barros, M. H., Carlson, C. G., Glerum, D. M., and Tzagoloff, A. (2001) Involvement of mitochondrial ferredoxin and Cox15p in hydroxylation of

heme O. FEBS Lett. 492, 133–138

 

 

Plasmids to facilitate mitochondrial purification

David Sabatini kindly gave permission for these to be listed here

3XHA-EGFP-OMP25

3XMyc-EGFP-OMP25

 

Another way to separate mitochondrial from cytosolic fractions used by some of our researchers, is use of the ThermoScientific Mitochondrial Isolation Kit for Mammalian Cells.