B6.Cg-Hbb^{tm2(HBG1,HBB*)Tow}/Hbb^{tm3(HBG1,HBB)Tow} Hba^tm1(HBA)Tow/ShmtJ

Stock number: 027265
Product name: hα/hα::β^A/β^S
Strain synonyms: Townes model
Research areas: Developmental Biology Research, Hematological Research, Internal/Organ Research

Description

Townes model mice carry several human hemoglobin knock-in genes replacing the endogenous mouse genes and may be useful in studying sickle cell disease.

Detailed description

In an attempt to offer alleles on well-characterized or multiple genetic backgrounds, alleles are frequently moved to a genetic background different from that on which an allele was first characterized. It should be noted that the phenotype of these C57BL/6J-congenic mice could vary from that originally described on a B6;129 genetic background (Stock No. 013071). We may modify the strain description if necessary as published results become available.

These mice harbor a combination of the following knock-in mutations:

1) the Hba^tm1(HBA)Tow mutation (also called hα ; replaces the endogenous mouse α-globin with the human α-globin gene), which is necessary to form the hemoglobin heterotetramer in conjunction with:

2) the Hbb^{tm2(HBG1,HBB*)Tow} mutation (also called -1400 γ-β^S ; replaces the endogenous mouse major and minor &beta-globin with the human hemoglobin gamma (Aγ) gene and the human sickle cell hemoglobin beta (β^S) gene),

3) the Hbb^{tm3(HBG1,HBB)Tow} mutation (also called -383 γ-β^A ; replacing the endogenous mouse major and minor β-globin with the human hemoglobin gamma (^Aγ) gene and the human wildtype hemoglobin beta (β^A) gene).

Mice homozygous for Hba^tm1(HBA)Tow and homozygous for Hbb^{tm2(HBG1,HBB*)Tow} have the sickle cell disease genotype and are referred to as hα/hα::β^S/β^S. This genotype on the C57BL/6J-congenic background is not viable - see below.

Mice homozygous for Hba^tm1(HBA)Tow and homozygous for Hbb^{tm3(HBG1,HBB)Tow} are viable, fertile and phenotypically normal. These mice are referred to as hα/hα::β^A/β^A and may be used as controls.

The presence of one copy of the Hbb^{tm3(HBG1,HBB)Tow} allele is sufficient to rescue the sickle cell phenotype. Therefore, mice homozygous for Hba^tm1(HBA)Tow and heterozygous for Hbb^{tm2(HBG1,HBB*)Tow} and Hbb^{tm3(HBG1,HBB)Tow} (compound heterozygous) are also viable, fertile and phenotypically normal and may be used for breeding, or as controls. These mice are referred to as hα/hα::β^A/β^S.

*Of note, none of the animals offered from this strain carry the mouse wildtype allele at either the Hba or Hbb loci*

In 2018, neither the donating laboratory nor The Jackson Laboratory had been able to generate C57BL/6J-congenic hα/hα::β^S/β^S live mice. Therefore, Stock No. 027265 is not viable as hα/hα::β^S/β^S. This is noteworthy as the same genotype is viable and exhibits the sickle cell disease phenotype on a B6;129 mixed genetic background (see Stock No. 013071).

Development

These mice harbor the hα (Hba^tm1(HBA)Tow) targeted mutation, the -1400 γ-β^S (Hbb^{tm2(HBG1,HBB*)Tow}) targeted mutation, and/or the -383 γ-β^A (Hbb^{tm3(HBG1,HBB)Tow}) targeted mutation. These mice should not harbor any wildtype allele at the Hbb locus.

The Hba^tm1(HBA)Tow targeted mutation (hα) was created by replacing the endogenous mouse α-globin gene with a human α-globin gene.

The Hbb^{tm2(HBG1,HBB*)Tow} targeted mutation (-1400 γ-β^S) was created by replacing the endogenous mouse major and minor β-globin genes with both the human ^Aγ gene (with 1400 bp of 5' flanking sequence) and the human β^S-globin gene (-1400 γ-β^S). The β^S gene contains an A to T mutation in the sixth codon of the human β-globin gene (resulting in a glutamic acid to valine conversion) that has been linked to sickle cell disease.

The Hbb^{tm3(HBG1,HBB)Tow} targeted mutation (-383 γ-β^A) was created by designing a targeting construct with the human hemoglobin gamma (^Aγ) gene with 383 bp of 5' flanking sequence, the human wildtype hemoglobin beta (β^A) gene with 815 bp of 5' flanking sequence, and a loxP-flanked phosphoglycerase/Hygromycin (PGK/Hygro) selection cassette.

The -383 γ-β^A targeting construct was electroporated into embryonic stem (ES) cells from knock-in sickle cell mice (homozygous for the hα targeted mutation and homozygous for the -1400 γ-β^S targeted mutation). The -383 γ-β^A construct contains 383 bp of 5' flanking sequence which is targeted to 1400 bp of 5' flanking sequence already present in the ES cells as part of the -1400 γ- β^S allele. Different recombination events in this flanking region produced two ES cell clones; clone 2 harbored the -383 γ-β^A replacement allele. This clone was then transiently transfected with a Cre expressing plasmid to delete the PGK/Hygro cassette, and then injected into recipient blastocysts. The resulting chimeric males were bred to females homozygous for hα and heterozygous for -1400 γ-β^S. Offspring with the sickle cell corrected genotypes (homozygous for hα and heterozygous at the Hbb locus (-383 γ-β^A/-1400 γ-β^S)) were obtained. These mice were maintained on a mixed C57BL/6;129 genetic background by breeding together for many generations prior to sending to The Jackson Laboratory Repository as Stock No. 013071.

Dr Paul Schmidt, from Boston Children's Hospital, purchased some Stock No. 013071 mice and subsequently separated the three alleles in order to backcross the strain to C57BL/6J (Stock No. 000664). Each allele was bred to C57BL/6J for at least 10 generations before being bred back together in one strain. The C57BL/6J-congenic strain was sent to The Jackson Laboratory as Stock No. 027265.

Allele

Symbol: Hbb^{tm2(HBG1,HBB*)Tow}
Name: targeted mutation 2, Timothy Townes
MGI accession ID: MGI:3790753
Generation method: Targeted
Attributes: Inserted expressed sequence; Humanized sequence
Marker symbol: Hbb
Marker name: hemoglobin beta chain complex
Chromosome: 7
Strain of origin: Not Specified
Expressed genes: HBG1,hemoglobin subunit gamma 1,human; HBB,hemoglobin subunit beta,human
Molecular note: The major and minor beta chains were replaced with a construct made of a 5.66 kb fragment of the human gamma chain including 1400 bp of 5' flanking sequence and a 4.1 kb fragment of the sickle cell form of human HBB in ES cells already containing a replacement of Hba complex with human HBA, Hba^tm1(HBA)Tow. Cre-mediated recombination removed the floxed hygro cassette.

Allele

Symbol: Hbb^{tm3(HBG1,HBB)Tow}
Name: targeted mutation 3, Timothy Townes
MGI accession ID: MGI:3790756
Generation method: Targeted
Attributes: Inserted expressed sequence; Humanized sequence
Marker symbol: Hbb
Marker name: hemoglobin beta chain complex
Chromosome: 7
Strain of origin: Not Specified
Expressed genes: HBG1,hemoglobin subunit gamma 1,human; HBB,hemoglobin subunit beta,human
Molecular note: In ES cells that were homozygous for Hba^tm1(HBA)Tow and Hbb^{tm2(HBG1,HBB*)Tow} the human gamma chain and beta chain containing the sickle cell mutation (an A to T transversion in the sixth codon) were replaced with the human gamma chain, beta chain (lacking the sickle cell mutation) and 383 bp of sequence flanking the gamma chain. Cre-mediated recombination removed the floxed hygro cassette.

Allele

Symbol: Hba^tm1(HBA)Tow
Name: targeted mutation 1, Timothy Townes
MGI accession ID: MGI:3790755
Generation method: Targeted
Attributes: Inserted expressed sequence; Humanized sequence
Marker symbol: Hba
Marker name: hemoglobin alpha chain complex
Chromosome: 11
Strain of origin: Not Specified
Expressed genes: HBA,hemoglobin, alpha,human
Molecular note: The complex was replaced with human HBA. Details will be described elsewhere.