Examples for targeting constructs


Preparation of a complex targeting construct to obtain a mouse model for the human disease “Seckel syndrome”

Murga M, Bunting S, Montaña MF, Soria R, Mulero F, Cañamero M, Lee Y, McKinnon PJ, Nussenzweig A and Fernandez-Capetillo O, 2009, A mouse model of ATR-Seckel shows embryonic replicative stress and accelerated aging, Nature Genetics, 41, 891

Using Red/ET recombination a targeting construct for a model of “Seckel” syndrome characterized by a severe deficiency in ATR was generated. Usually ATR kinase (Ataxia Telangiectasia and Rad3-related serine/threonine-protein kinase) prevents replicative stress, a source of endogenous DNA damage. A point mutation in exon 9 was thought to be responsible for an incorrect slicing of the mRNA, which results in a deletion of exon 9. The animal model based on the constructs given below confirmed this hypothesis.

Cloning strategy: 1. Replacement of entire mouse genomic fragment encompassing exon 8, 9 and 10 of ATR gene, with human counterpart; 2. Introduction of “Seckel” mutation into humanized mouse allele (nucleotide exchange from adenine to guanine)

Final targeting construct for ES cell recombination generated by Gene Bridges

Analysis of the mouse model: “Seckel” mice show high levels of replicative stress during embryogenesis, when proliferation is widespread, but this is reduced to marginal amounts in postnatal life. In spite of this decrease, adult “Seckel” mice show accelerated aging, which is further aggravated in the absence of p53. Together, these results support a model whereby replicative stress, particularly in utero, contributes to the onset of aging in postnatal life, and this is balanced by the replicative stress–limiting role of the checkpoint proteins ATR and p53.


Preparation of a BDNF-BAC transgene by fusing the 10 kb full-length huntigtin cDNA under the control of the BDNF promoter to obtain a new animal model for Huntington’s Disease

Hager S, Lösch S, Noll S, Khan-Vaughan L, Ehrlich ME and Kranz H, 2012, Red/ET recombination with chimeric oligonucleotides allows rapid generation of BAC transgene harboring full-length or truncated huntingtin cDNA, BioTechniques Rapid Dispatches, doi: 10.2144/000113908

Huntington's disease (HD) is a fatal neurodegenerative disorder, which is caused by a CAG repeat expansion encoding a poly-glutamine tract in the huntingtin (htt) gene. The recapitulation of the exact disease symptoms and course is rather difficult, since the generation of HD disease (mouse) models is challenging because of the size and complexity of the htt gene locus. Using recombineering, four novel BAC transgenes harboring full-length or truncated htt cDNA comprising 98 or 15 glutamine residues were generated, which will further research into Huntington's disease.

Therefore the notoriously difficult huntingtin cDNA was transferred into the heterologous genomic locus BDNF (brain-derived neurotrophic factor). BDNF is expressed in the cortical neurons projecting to the striatal medium spiny neurons, and was used to direct htt transgene expression to investigate the contribution of these cell types to HD.



Cloning strategy: Using three Red/ET recombination steps and a final FLP recombination huntingtin cDNA was transferred into the BDNF locus of a suitable BAC. Chimeric oligonucleotides comprising a primer binding site and a bifunctional homology arm were used in PCR-amplification of antibiotic resistance cassettes which were inserted into the substrate plasmid adjacent to the htt cDNA. Using unique restriction sites, the cDNA was excised from the source plasmid and transferred into the BAC.

The recombineering protocol enabled the generation of four mouse models of HD and is applicable to many other complex cloning exercises too.


Construction of chimeric human-rat IgH loci by BAC stitching and counterselection

Ma B, Osborn MJ, Avis S, Ouisse LH, Mènoret S, Anegon I, Buelow R and Brüggemann M, 2013, Human antibody expression in transgenic rat: Comparison of chimeric IgH loci with human VH, D and JH but bearing different rat C-gene regions, J of Immunological Methods, 400-401, 78

Gene Bridges generated chimeric human-rat IgH loci using the Red/ET recombination technology for human antibody expression studies in transgenic rats. Therefore a 130 kb construct was assembled by combining DNA fragments of interest from three different BACs coding for the rat IgH locus by several Red/ET recombination steps (BAC stitching). Finally a part of the fused DNA was humanized by exchanging a 2 kb domain via a counter selection by Red/ET recombineering.

Scheme of BAC stitching and final humanizing of a 130 kb construct

To the top

Please choose your country. If the country is missing, continue.

Shanghai QF Biosciences
Shanghai QF Biosciences
NeuProCell
Biotech Desk
NeuProCell
Biotech Desk
BioTag
BioTag
Funakoshi
Funakoshi
Mbiotech
Mbiotech
iDNA
iDNA
iDNA
iDNA
iDNA
iDNA
iDNA
iDNA
Gendiscovery
Gendiscovery
BioCat
BioCat
BioCat
BioCat
BioCat
BioCat
Biovalley
Biovalley
Quimigen
Quimigen
Brunschwig Chemie
Brunschwig Chemie
Cambio
Cambio
Cambio
Cambio
Immuno Diagnostic
Immuno Diagnostic
Immuno Diagnostic
Immuno Diagnostic
Immuno Diagnostic
Immuno Diagnostic
Immuno Diagnostic
Immuno Diagnostic
Immuno Diagnostic
Nanodiagnostika
Immuno Diagnostic
Nanodiagnostika
Nanodiagnostika
Nanodiagnostika
Nanodiagnostika
Nanodiagnostika
AS ONE INTERNATIONAL, INC.
AS ONE INTERNATIONAL, INC.
Valquim
Valquim