Generation of Knock-Out lines for actin cytoskeleton regulators (FCHSD2 and FGD2), and their role in B cells
Vinod, Rufus (2019-03-06)
Generation of Knock-Out lines for actin cytoskeleton regulators (FCHSD2 and FGD2), and their role in B cells
(06.03.2019)
Lataukset:
Julkaisu on tekijänoikeussäännösten alainen. Teosta voi lukea ja tulostaa henkilökohtaista käyttöä varten. Käyttö kaupallisiin tarkoituksiin on kielletty.
suljettu
Julkaisun pysyvä osoite on:
https://urn.fi/URN:NBN:fi-fe2019032810305
https://urn.fi/URN:NBN:fi-fe2019032810305
Tiivistelmä
B lymphocytes are cells of the immune system that produce variety of antibodies. Antigen binding to B cell receptor (BCR) leads to transmembrane signaling and antigen internalization. B cell presents internalized antigen to CD4+ helper T cells and receive co-stimulatory signal within the structure called immunological synapse. These steps are crucial for the development of protective antibody response and rely on tightly regulated action of actin cytoskeleton system. The key role in actin cytoskeleton regulation is played small GTPases of the Rho family (Rac1, Cdc42, RhoA) regulated by guanine nucleotide exchange factors (GEFs). BAR domain family of proteins recruit actin cytoskeleton regulatory proteins to cellular membranes. BAR-domain protein FCHSD2 plays a significant role in membrane remodeling and actin polymerization, whereas FGD2 has RhoGEF activity towards Cdc42.
To understand the function of FCHSD2 and FGD2 in B cells, their corresponding genes were targeted using CRISPR/Cas9 system. Guide RNA (gRNA) sequences for targeted deletion of two genomic regions in each gene were cloned into plasmids containing Cas9-GFP. Plasmids were electroporated into A20 D1.3 B cells and the ones expressing GFP were enriched by cell sorting. Clones with gene deletions were selected by PCR analysis and lack of protein expression was validated by immunoblotting. Antigen presentation was studied by co-culture of generated B cell lines with 1E5 hybridoma T cells followed by measurements of IL-2 production in the culture supernatants by ELISA. BCR internalization and expression of surface molecules was assessed by flow cytometry.
We successfully generated knockout (KO) and heterozygote (HEZ) clones for proteins. The lack of FCHSD2 expression was confirmed by immunoblotting, however, FGD2 deletion could not be verified due to lack of functional antibodies. Antigen presentation assay showed that IL-2 production by T cells was reduced over 3-fold upon co-culture with FCHSD2 KO B cells. We also found that FCHSD2 KO B cells are severely defected in their surface IgM expression, whereas expression of IgG2a (BCR), CD19 and B220 remained intact. Thus, we established FCHSD2 as previously uncharacterized protein essential for normal surface expression of IgM in mouse B lymphocytes.
To understand the function of FCHSD2 and FGD2 in B cells, their corresponding genes were targeted using CRISPR/Cas9 system. Guide RNA (gRNA) sequences for targeted deletion of two genomic regions in each gene were cloned into plasmids containing Cas9-GFP. Plasmids were electroporated into A20 D1.3 B cells and the ones expressing GFP were enriched by cell sorting. Clones with gene deletions were selected by PCR analysis and lack of protein expression was validated by immunoblotting. Antigen presentation was studied by co-culture of generated B cell lines with 1E5 hybridoma T cells followed by measurements of IL-2 production in the culture supernatants by ELISA. BCR internalization and expression of surface molecules was assessed by flow cytometry.
We successfully generated knockout (KO) and heterozygote (HEZ) clones for proteins. The lack of FCHSD2 expression was confirmed by immunoblotting, however, FGD2 deletion could not be verified due to lack of functional antibodies. Antigen presentation assay showed that IL-2 production by T cells was reduced over 3-fold upon co-culture with FCHSD2 KO B cells. We also found that FCHSD2 KO B cells are severely defected in their surface IgM expression, whereas expression of IgG2a (BCR), CD19 and B220 remained intact. Thus, we established FCHSD2 as previously uncharacterized protein essential for normal surface expression of IgM in mouse B lymphocytes.