Genome Editing of Erythroid Cell Culture Model Systems

Yik, Jinfen J.; Crossley, Merlin; Quinlan, Kate

doi:10.1007/978-1-4939-7428-3_15

Cited by 3 publications

(6 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We showed previously that Tmod3 is required for terminal differentiation of mouse fetal liver definitive erythroblasts in vivo , but whether this was a consequence of Tmod3 regulation of F-actin was not explored (16). To directly examine the role of Tmod3 in ED, we adopted a CRISPR-Cas9 mediated knockout methodology to perturb the Tmod3 gene in a mouse erythroleukemia cell line, Mel ds19 (19). We directed disruption of Tmod3 gene exon 9b via a sgRNA-Cas9-GFP construct leading to a double-strand break followed by canonical non-homology end joining (NHEJ) directed repair (Figure 1A).…”

Section: Resultsmentioning

confidence: 99%

“…Here, to directly assess the role of Tmod3 in ED and membrane skeleton assembly we employed a murine erythroleukemia cell line (Mel ds19), which has been widely used to investigate many erythroid processes, including ED and membrane skeleton assembly, and is amenable to efficient and rapid genetic manipulation (18, 19). We generated a Tmod3 knockout Mel cell line using CRISPR-Cas9 technology and compared ED induced by DMSO in the parental Mel ds19 cells with Tmod3-knockout Mel cells.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Erythroid differentiation in mouse erythroleukemia cells is driven via actin filament-tropomodulin3-tropomyosin networks

Ghosh

Coffin

West

et al. 2021

Preprint

View full text Add to dashboard Cite

Erythroid differentiation (ED) is a complex cellular process entailing morphologically distinct maturation stages of erythroblasts during terminal differentiation. Studies of actin filament assembly and organization during terminal ED have revealed essential roles for the pointed-end actin filament capping proteins, tropomodulins (Tmod1 and Tmod3). Additionally, tropomyosin (Tpm) binding to Tmods is a key feature promoting Tmod-mediated actin filament capping. Global deletion of Tmod3 leads to embryonic lethality in mice with impaired ED. To test a cell autonomous function for Tmod3 and further decipher its biochemical function during ED, we generated a Tmod3 knockout in a mouse erythroleukemia cell line (Mel ds19). Tmod3 knockout cells appeared normal prior to ED, but showed defects during progression of ED, characterized by a marked failure to reduce cell and nuclear size, reduced viability and increased apoptosis. In Mel ds19 cells, both Tpms and actin were preferentially associated with the Triton-X 100 insoluble cytoskeleton during ED, indicating Tpm-coated actin filament assembly during ED. While loss of Tmod3 did not lead to a change in total actin levels, it led to a severe reduction in the proportion of Tpms and actin associated with the Triton-X 100 insoluble cytoskeleton during ED. We conclude that Tmod3-regulation of actin cytoskeleton assembly via Tpms is integral to morphological maturation and cell survival during normal erythroid terminal differentiation.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Erythroid differentiation in mouse erythroleukemia cells is driven via actin filament-tropomodulin3-tropomyosin networks

Ghosh

Coffin

West

et al. 2021

Preprint

View full text Add to dashboard Cite

show abstract

“…PX458 (pSpCas[BB]‐2A‐GFP) plasmid was a gift from Feng Zhang (Addgene plasmid no. 48138), and the protocol of CRISPR/Cas9 point mutation was adopted from Yik et al 25 Five micrograms of PX458 was digested with Bbs I (25 U) with New England Biolabs (Ipswich, MA) buffer 2.1 in a total volume of 40 µL, whereby digestion reaction was performed by incubating at 37°C for 1 hour. Bbs I was heat inactivated at 65°C for 20 minutes before dephosphorylation by CIP (5 U) (New England Biolabs) at 37°C for 1 hour.…”

Section: Methodsmentioning

confidence: 99%

Serine 26 in Early Growth Response‐1 Is Critical for Endothelial Proliferation, Migration, and Network Formation

Santiago

Khachigian

2021

JAHA

View full text Add to dashboard Cite

Background Vascular endothelial cell proliferation, migration, and network formation are key proangiogenic processes involving the prototypic immediate early gene product, Egr‐1 (early growth response‐1). Egr‐1 undergoes phosphorylation at a conserved Ser26 but its function is completely unknown in endothelial cells or any other cell type. Methods and Results A CRISPR/Cas9 strategy was used to introduce a homozygous Ser26>Ala mutation into endogenous Egr‐1 in human microvascular endothelial cells. In the course of generating mutant cells, we produced cells with homozygous deletion in Egr ‐1 caused by frameshift and premature termination. We found that Ser26 mutation in Egr‐1, or Egr‐1 deletion, perturbed endothelial cell proliferation in models of cell counting or real‐time growth using the xCELLigence System. We found that Ser26 mutation or Egr‐1 deletion ameliorated endothelial cell migration toward VEGF‐A 165 (vascular endothelial growth factor‐A) in a dual‐chamber model. On solubilized basement membrane preparations, Ser26 mutation or Egr‐1 deletion prevented endothelial network (or tubule) formation, an in vitro model of angiogenesis. Flow cytometry further revealed that Ser26 mutation or Egr‐1 deletion elevated early and late apoptosis. Finally, we demonstrated that Ser26 mutation or Egr‐1 deletion increased VE‐cadherin (vascular endothelial cadherin) expression, a regulator of endothelial adhesion and signaling, permeability, and angiogenesis. Conclusions These findings not only indicate that Egr‐1 is essential for endothelial cell proliferation, migration, and network formation, but also show that point mutation in Ser26 is sufficient to impair each of these processes and trigger apoptosis as effectively as the absence of Egr‐1. This highlights the importance of Ser26 in Egr‐1 for a range of proangiogenic processes.

show abstract

Section: Introductionmentioning

confidence: 99%

“…Here, to directly assess the role of Tmod3 in ED and membrane skeleton assembly we employed a murine erythroleukemia cell line (Mel ds19), which has been widely used to investigate many erythroid processes, including ED and membrane skeleton assembly, and is amenable to efficient and rapid genetic manipulation. 25 We generated a Tmod3 knockout Mel cell line using CRISPR-Cas9 technology and compared ED induced by DMSO in the parental Mel ds19 cells with Tmod3-knockout Mel cells. We utilized standard Triton X-100 based extraction and centrifugation to separate soluble and insoluble fractions to empirically assess membrane skeleton assembly in these cells.…”

Section: Introductionmentioning

confidence: 99%

Erythroid differentiation in mouse erythroleukemia cells depends on Tmod3‐mediated regulation of actin filament assembly into the erythroblast membrane skeleton

et al. 2022

View full text Add to dashboard Cite

Erythroid differentiation (ED) is a complex cellular process entailing morphologically distinct maturation stages of erythroblasts during terminal differentiation. Studies of actin filament (F‐actin) assembly and organization during terminal ED have revealed essential roles for the F‐actin pointed‐end capping proteins, tropomodulins (Tmod1 and Tmod3). Tmods bind tropomyosins (Tpms), which enhance Tmod capping and F‐actin stabilization. Tmods can also nucleate F‐actin assembly, independent of Tpms. Tmod1 is present in the red blood cell (RBC) membrane skeleton, and deletion of Tmod1 in mice leads to a mild compensated anemia due to mis‐regulated F‐actin lengths and membrane instability. Tmod3 is not present in RBCs, and global deletion of Tmod3 leads to embryonic lethality in mice with impaired ED. To further decipher Tmod3’s function during ED, we generated a Tmod3 knockout in a mouse erythroleukemia cell line (Mel ds19). Tmod3 knockout cells appeared normal prior to ED, but showed defects during progression of ED, characterized by a marked failure to reduce cell and nuclear size, reduced viability, and increased apoptosis. Tmod3 does not assemble with Tmod1 and Tpms into the Triton X‐100 insoluble membrane skeleton during ED, and loss of Tmod3 had no effect on α1,β1‐spectrin and protein 4.1R assembly into the membrane skeleton. However, F‐actin, Tmod1 and Tpms failed to assemble into the membrane skeleton during ED in absence of Tmod3. We propose that Tmod3 nucleation of F‐actin assembly promotes incorporation of Tmod1 and Tpms into membrane skeleton F‐actin, and that this is integral to morphological maturation and cell survival during erythroid terminal differentiation.

show abstract

Genome Editing of Erythroid Cell Culture Model Systems

Cited by 3 publications

References 20 publications

Erythroid differentiation in mouse erythroleukemia cells is driven via actin filament-tropomodulin3-tropomyosin networks

Erythroid differentiation in mouse erythroleukemia cells is driven via actin filament-tropomodulin3-tropomyosin networks

Serine 26 in Early Growth Response‐1 Is Critical for Endothelial Proliferation, Migration, and Network Formation

Erythroid differentiation in mouse erythroleukemia cells depends on Tmod3‐mediated regulation of actin filament assembly into the erythroblast membrane skeleton

Contact Info

Product

Resources

About