4B6/3598/3442#2 Cell Line

This murine fibroblast cell line is a derivative of the 4B6 cell line (Cat. ESA102). Unlike the parental cell line, it expresses the Escherichia coli Lig A gene in place of the endogenous DNA ligase III. To generate 4B6/3598/3442#2 cells, 4B6 cell line was transduced with retroviruses rv3598 (G418 resistance) and rv3442 (puromycin resistance) resulting in the inactivation of the cellular Lig 3 gene through Cre-mediated excision (rv3442) and re-expression of the bacterial LigA gene devoid of mitochondrial targeting signal (rv3598). Inefficient mitochondrial import of the LigA in the absence of the Lig 3 makes DNA ligase activity limiting for the mtDNA replication, and leads to a reduced (10-100 fold) mtDNA copy number.

From the laboratory of Mikhail F Alexeyev, PhD, University of South Alabama.

Catalog Number Product Size AVAILABILITY Price Qty
ESA103
4B6/3598/3442#2 Cell Line
1 vial 3-5 weeks
Price: $399.00
Specifications
Product Type: Cell Line
Cell Type: Embryonic fibroblasts
Organism: Mouse
Source: Mouse E13.5 Embryo
Morphology: Spindle-shaped at confluence
Biosafety Level: BSL 1
Subculturing: Split 1:4 every 3 days
Growth Conditions: DMEM/10% FBS. Periodic selection with 10 µg/ml blasticidin, 2 µg/ml puromycin, and 1 mg/ml G418
Cryopreservation: DMEM/10% FBS/10% DMSO
Comments: Slow- Growing
Storage: LN2
Shipped: Dry Ice
Provider
From the laboratory of Mikhail F Alexeyev, PhD, University of South Alabama.

Comments
Cre-TM mice (JAX 004682, B6.Cg-Tg(CAG-cre/Esr1)5Amc/J) were intercrossed with Lig3LoxP mice (Nature 471(2011) 240-244) to provide tamoxifen-inducible cre expression driven from the actin promoter. Lig3Cre-TM mouse embryonic fibroblast (MEF) cell cultures were prepared from E13.5 embryonic mesenchyme. Cells were allowed to proliferate in T-25 tissue culture flasks at 37 °C in a humidified CO2-regulated (5%) incubator and were immortalized with a retroviral construct 3315, which encodes SV40 large T antigen (J. Biol. Chem. 288 (2013) 26594-26605) and selected depending upon their ability to form colonies. One clone was selected for its superior growth characteristics and designated Cre4. This clone was transduced with a retrovirus 2641 (Mol. Biol. Rep. 37 (2010) 1987-1991, which encodes a Tet-On Advanced transactivator, EGFP and blasticidin resistance), thus producing a clone designated 4B6. 4B6 is a Tet-On derivative of the Cre4. Cre4 were further transduced with retroviruses rv3598 (G418 resistance) and rv3442 (puromycin resistance) resulting in the inactivation of the cellular Lig 3 gene through Cre-mediated excision (rv3442) and re-expression of the bacterial LigA gene devoid of mitochondrial targeting signal. Inefficient mitochondrial import of the LigA makes DNA ligase activity limiting for the mtDNA replication, and leads to a reduced mtDNA copy number.
References
  1. Spadafora D, Kozhukhar N, Alexeyev MF. Presequence-Independent Mitochondrial Import of DNA Ligase Facilitates Establishment of Cell Lines with Reduced mtDNA Copy Number. PLoS One. 2016 Mar 31;11(3):e0152705.

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