Generating mKate2 Expression Under Xanf1 Promoter

To generate a construct expressing mKate2 under the control of Xanf1 promoter, the 2200 bp fragment of this promoter sufficient for specifically targeting the expression to the anterior neural plate [10 (link)] was subcloned into VspI (blunted)/AgeI sites of mKate2-N vector, instead of the multiple cloning site region and CMV (cytomegalovirus) promoter. For transgenic experiments, vectors were linearized by SfiI and purified using Qiagen columns. Transgenic embryos were generated by the REMI (restriction enzyme-mediated integration) technique exactly as described previously [10 (link)] and were analysed using a Leica MZFLIII fluorescent stereomicroscope with the rhodamine filter set: excitation filter 546/10 nm; suppression filter 565 nm. To visualize a fluorescent signal on tissue sections, transgenic embryos were fixed overnight in 4 % (w/v) formaldehyde in 0.1 % PBS, embedded in 3 % agarose in 0.1 % PBS and sectioned using a HB 650 Vibratome (Microme) in 50 μm sections. The animal work was performed in Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, in accordance with the regulations of the Department of Health and Human Services, National Institutes of Health.

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SHCHERBO D., MURPHY C.S., ERMAKOVA G.V., SOLOVIEVA E.A., CHEPURNYKH T.V., SHCHEGLOV A.S., VERKHUSHA V.V., PLETNEV V.Z., HAZELWOOD K.L., ROCHE P.M., LUKYANOV S., ZARAISKY A.G., DAVIDSON M.W, & CHUDAKOV D.M. (2009). Far-red fluorescent tags for protein imaging in living tissues. The Biochemical journal, 418(3), 567-574.

Publication 2009

Agarose Animal Cytomegalovirus Embryos Formaldehyde Neural plate Restriction enzyme Rhodamine Tissue Transgenic Vectors

Corresponding Organization :

Other organizations : Institute of Bioorganic Chemistry, National High Magnetic Field Laboratory, Florida State University, Albert Einstein College of Medicine

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SARS-CoV-2 Variant S-Protein Antigen Constructs

The S-protein antigen constructs representing the different VOCs contained the following mutations compared to the WT variant (Wuhan Hu-1; GenBank: MN908947.3): deletion (Δ) of H69, V70 and Y144, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H in Alpha (B.1.1.7); L18F, D80A, D215G, L242H, R246I, K417N, E484K, N501Y, D614G, and A701V in Beta (B.1.351); and L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, and T1027I in Gamma(P.1). The genes were ordered as gBlock gene fragments (Integrated DNA Technologies) and cloned Pst I/Not I in a pPPI4 expression vector containing a hexahistidine (his) tag with Gibson Assembly (Thermo Fisher Scientific). All S constructs were verified by Sanger sequencing and the protein was subsequently produced in human embryonic kidney (HEK) 293 F cells (Thermo Fisher Scientific) and purified as previously described^{4 (link),9 (link)}.

van Rijswijck D.M., Bondt A., Hoek M., van der Straten K., Caniels T.G., Poniman M., Eggink D., Reusken C., de Bree G.J., Sanders R.W., van Gils M.J, & Heck A.J. (2022). Discriminating cross-reactivity in polyclonal IgG1 responses against SARS-CoV-2 variants of concern. Nature Communications, 13, 6103.

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SARS-CoV-2 Spike Protein Variant Expression

The mutations compared to the WT variant (Wuhan Hu-1; GenBank: MN908947.3) in the S proteins are depicted in S1 Table. The S constructs were ordered as gBlock gene fragments (Integrated DNA Technologies) and cloned in a pPPI4 expression vector containing a hexahistidine (his) tag with Gibson Assembly (ThermoFisher) [32 (link)]. All S constructs were verified by Sanger sequencing, subsequently produced in HEK293F cells (ThermoFisher), and purified as previously described [32 (link)].

van Gils M.J., Lavell A., van der Straten K., Appelman B., Bontjer I., Poniman M., Burger J.A., Oomen M., Bouhuijs J.H., van Vught L.A., Slim M.A., Schinkel M., Wynberg E., van Willigen H.D., Grobben M., Tejjani K., van Rijswijk J., Snitselaar J.L., Caniels T.G., Vlaar A.P., Prins M., de Jong M.D., de Bree G.J., Sikkens J.J., Bomers M.K, & Sanders R.W. (2022). Antibody responses against SARS-CoV-2 variants induced by four different SARS-CoV-2 vaccines in health care workers in the Netherlands: A prospective cohort study. PLoS Medicine, 19(5), e1003991.

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Structural Analysis of SARS-CoV-2 Spike Variants

The 2P-stabilized S proteins
of the Wuhan strain (WT) and B.1.351
variant were described previously.^{12 (link),55 (link)} The B.1.351
construct contained the following mutations compared to the WT variant
(Wuhan Hu-1; GenBank: MN908947.3): L18F, D80A, D215G, L242H, R246I,
K417N, E484K, N501Y, D614G, and A701V. Both S constructs were produced
in HEK293F suspension cells (ThermoFisher) and purified as previously
described.^{12 (link)} For the human ACE2 receptor,
soluble ACE2 was generated as described previously^{12 (link)} by using a gene encoding amino acids 18–740 of ACE2.
The IgGs and Fab fragments used in this study were produced as previously
described.^{12 (link),26 (link)}

Yin V., Lai S.H., Caniels T.G., Brouwer P.J., Brinkkemper M., Aldon Y., Liu H., Yuan M., Wilson I.A., Sanders R.W., van Gils M.J, & Heck A.J. (2021). Probing Affinity, Avidity, Anticooperativity, and Competition in Antibody and Receptor Binding to the SARS-CoV-2 Spike by Single Particle Mass Analyses. ACS Central Science, 7(11), 1863-1873.

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Variable analysis

independent variables

Xanf1 promoter fragment

dependent variables

MKate2 expression

control variables

VspI (blunted)/AgeI sites of mKate2-N vector
Multiple cloning site region and CMV (cytomegalovirus) promoter
Linearization by SfiI restriction enzyme
REMI (restriction enzyme-mediated integration) technique

positive controls

Previous experiments as described in reference [10 (https://pubmed.ncbi.nlm.nih.gov/15128667/)]

negative controls

Not explicitly mentioned

Annotations

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