4B6/3598/3442#2/4011/4014C1-2 Cell Line

This murine fibroblast cell line is a derivative of the 4B6/3598/3442#2/4011C1 Cell Line (Cat. ESA104) and maintains mtDNA at a reduced copy number. To generate this cell line the parental cells, which maintain mtDNA copy number at normal levels were transiently transfected with a plasmid pMA4014, which encodes mCherry and FLPo and flow sorted for transfected cells. As a result of FLPo-mediated excision of the MTS in front of the bacterial LigA gene, mitochondrial import of LigA in 4B6/3598/3442#2/4011/4014C1-2 cells is inefficient, and mtDNA is maintained at a reduced copy number. This cell line in conjunction with its companion isogenic cell line 4B6/3598/3442#2/4011C1 (Cat. ESA104) is useful for studying the effects of mtDNA copy number on cellular physiology.

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

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4B6/3598/3442#2/4011/4014C1-2 Cell Line
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Product Type: Cell Line
Cell Type: Embryonic fibroblasts
Morphology: Spindle-shaped at confluence
Organism: Mouse
Source: Mouse E13.5 Embryo
Biosafety Level: BSL 1
Growth Conditions: DMEM/10% FBS. Periodic selection with 10 µg/ml blasticidin, 2 µg/ml puromycin, and 1 mg/ml G418
Subculturing: Split 1:4 to 1:8 every 3 days
Cryopreservation: DMEM/10% FBS/10% DMSO
Comments: Fast Growing
Storage: LN2
Shipped: Dry Ice

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

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 immortalized with a retroviral construct 3315, which encodes SV40 large T antigen (J. Biol. Chem. 288 (2013) 26594-26605) and selected depending based on 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 (MTS, rv3598). Inefficient mitochondrial import of the LigA makes DNA ligase activity in the absence of the Lig 3 limiting for the mtDNA replication and leads to a reduced mtDNA copy number. These cells were transduced with a retrovirus rv4011, which encodes the same LigA fused to an excisable (FLPo) mitochondrial targeting sequence from human ornithine transcarbamylase. This fusion protein is efficiently imported into mitochondria, and therefore normal mtDNA copy number is restored in the transduced cells. Finally, the resulting cells were transiently transfected with a plasmid 4014, which encodes mCherry and FLPo. As a consequence of the FLPo-mediated removal of the MTS from LigA, mtDNA copy number in the resulting clone #4B6/3598/3442#2/4011/4014C1-2 is reduced. This cell line, in conjunction with an isogenic cell line 4B6/3598/3442#2/4011C1 is useful for studying effects of mtDNA copy number on cellular physiology.
  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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