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

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

This murine fibroblast cell line is a derivative of the 4B6/3598/3442#2 Cell Line (Cat. ESA103). Unlike the parental cell line, these cells maintain mtDNA at normal levels. This was achieved by introducing into 4B6/3598/3442#2 cells of a LigA gene fused to an excisable (FRT/FLPo) mitochondrial targeting sequence (MTS) sequence from human ornithine transcarbamylase (rv4011). The resulting cells express two LigA variants: one without- and another with MTS. This latter (with MTS) variant is efficiently imported into mitochondria, and therefore normal mtDNA copy number is restored in the transduced cells.

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

Catalog Number Product DataSheet Size AVAILABILITY Price Qty
ESA104
4B6/3598/3442#2/4011C1 Cell Line
1 vial In stock
Regular Price:$460.00
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Specifications

Product Type: Cell Line
Cell Type: Embryonic fibroblasts
Source: Mouse E13.5 Embryo
Organism: Mouse
Morphology: Spindle-shaped at confluence
Biosafety Level: BSL 1
Subculturing: Split 1:4 to 1:8 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: Media + 10% DMSO
Comments: Fast 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 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 (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. However, FLPo-mediated excision of the MTS reduces the efficiency of the mitochondrial import of the LigA and results in the reduced mtDNA copy number (as in the companion cell line 4B6/3598/3442#2/4011C1-2). This cell line in conjunction with its companion isogenic cell line 4B6/3598/3442#2/4011C1-2 is useful for studying effects of mtDNA copy number on cellular physiology.
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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