CONTACT FOR REAGENT AND RESOURCE SHARING
Further information and requests for reagents may be directed to, and will be fulfilled by the Lead Contact, Frederick Alt (alt@enders.
tch.harvard.edu).
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Mice
The VH1-2 and VH1-2/LC mouse models were generated and housed in the animal facility at Boston Children’s Hospital. The animal
work was covered under protocol 14-10-2790R, which was approved by Institutional Animal Care and Use Committee of Boston
Children’s Hospital. The VH1-2 mouse model was derived from a F1 hybrid (129/Sv: C57BL/6) ES clone in which human IGHV1-
2*02 replaced the mouse VH81X, and IGCRI was deleted on theIgHaallele (129/Sv) (See text andMethods Details). The ES clone
was injected into Rag2/blastocysts to generate RDBC chimeras. The chimeric mice were bred with 129/Sv mice for germline
transmission of the targeted mutations. Heterozygous VH1-2/IGCRI mice were bred to homozygosity (VH1-2 mouse model). In
VH1-2 mouse model, both IgH alleles are of IgHaallotype (129/Sv), but the other chromosomes are of mixed 129/Sv and C57BL/6
origins. The VH1-2/LC mouse model was derived from the F1 ES clone described above that contained the VH1-2 replacement
and IGCRI deletion by further gene targeted replacement of the mouseJklocus with the precursor VRC01 light chain and targeted
deletion of theJHballele (See text andMethods Details). The resultant ES clone was injected into Rag2/blastocysts to generate
chimeric mice (VH1-2/LC mouse model), which are of mixed 129/Sv and C57BL/6 genetic background.
For immunization studies, the VH1-2 and VH1-2/LC mice were housed and cared for in accordance with local, state, federal, and
institute policies in an American Association for Accreditation of Laboratory Animal Care-accredited facility at the NIH. All animal
experiments were reviewed and approved by the Animal Care and Use Committee of the Vaccine Research Center, NIAID, NIH.
The animal work was covered under protocol VRC 14-467 for breeding, and VRC 14-480 for immunization of mice.
METHODS DETAILS
Generation and Characterization of VH1-2 and VH1-2/LC Mouse Models
All the genetic modifications were introduced into ES cells, using standard gene targeting technology (e.g.,Chen et al., 1993; Guo
et al., 2011). To illustrate the method of genetic modification, the replacement of VH81X with IGHV1-202 is described here as an
example; all the other genetic modifications followed the same procedure. The targeting construct was based on PGKneolox2DTA.2
(Addgene #13449). In this targeting construct, the IGHV1-202 segment, from the start codon in leader exon to the end of VHexon, is
Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Human: Expi293F cells ThermoFisher Scientific Cat# A14528
Human: FreeStyle 293F cells ThermoFisher Scientific Cat# R79007
Experimental Models: Organisms/Strains
Mus musculus:VH1-2 mouse model; mixed
129/Sv and C57BL/6 strains
This paper N/AMus musculus:VH1-2/LC mouse model;
mixed 129/Sv and C57BL/6 strains
This paper N/ASequence-Based Reagents
Primers for single cell RT-PCR in the VH1-2
mouse model and the VH1-2/LC mouse
models
This paper Table S6Paired heavy and light chain emulsion
linkage RT-PCR primer mix and nested
PCR primer mix for amplification of linked
VH:VL constructs
This paper Table S7Software and Algorithms
HTGTS-rep-seq Lin et al., 2016 N/A
GraphPad Prism 6.05 Software GraphPad Prism Software
MUSCLE Edgar, 2004 http://www.ebi.ac.uk/Tools/msa/muscle/
SeaView Gouy et al., 2010 http://doua.prabi.fr/software/seaview
IMGT/V-Quest IMGT http://www.imgt.org/IMGT_vquest/vquest
e4 Cell 166 , 1471–1484.e1–e8, September 8, 2016