Point Mutation Knockin Mouse Models
A point mutation Knockin mouse defines an animal model in which one or more nucleotides are constitutively mutated.
The insertion, deletion, nonsense and sense mutations can alter the amino acid sequence of a given protein, and so dramatically affect its function.
Typical applications for point mutation Knockin mouse models
For academic research:
For bio-pharmaceutical research & development:
Strengths and limitations of point mutation Knockin mouse models
- Best way to reproduce human disease when due to mutations
- High physiological relevancy of the scientific data obtained from the model (regulatory elements conserved, under control of endogenous promoter, expression of all splice variants, etc.) = cleaner way than classical KO where the whole gene is deleted
- Phenotype due only to the mutation: alteration of a single function without disturbing other domains of a protein
- Mutation of the gene of interest may affect development, resulting in an impaired phenotype or embryonic death
→ Limitation can be bypassed by applying conditions such as time-specific gene inactivation
- 1. Modification or disruption of splicing regulation
2. Genetic redundancy
→ Can be assessed via constitutive Knockout of the gene of interest
Case studies and publications on our point mutation Knockin mouse models
Model with heterozygous point mutations, exactly homologous to human retinitis pigmentosa (RP).
Sothilingam V, et al. Retinitis pigmentosa: impact of different Pde6a point mutations on the disease phenotype. Hum Mol Genet. 2015.
Mutations in the PDE6A gene can cause rod photoreceptor degeneration and the blinding disease retinitis pigmentosa.
Model: Novel mouse model for the Pde6aR562W point mutation in combination with an existing line carrying the V685M point mutation to generate compound heterozygous Pde6aV685M/R562W animals, exactly homologous to a case of human RP.
Aim: Predict time-courses for Pde6a-related retinal degeneration and thereby facilitate the definition of a window of opportunity for clinical interventions.
Results: The study provides a rational basis for predictions on human RP phenotypes and disease progression in compound heterozygous situations, and suggests the targeting of non-apoptotic processes as a feasible treatment approach.
Figure 1. Loss of PDE6A expression causes cGMP accumulation.
Excessive accumulation of cGMP and subsequent rod photoreceptor death, followed by a mutation-independent, secondary death of cone photoreceptors.
A, B) In the PN11 wildtype (wt) retina (A), immunostaining for PDE6A shows strong protein expression in photoreceptor OS.
In contrast, at the same age, in the compound heterozygous V685M/R562W retina (C), the protein is essentially absent.
C, D) At PN11, wt retina is essentially negative for cGMP immunoreactivity (C).
PDE6A mutants, however, display individual rod photoreceptor cells that have accumulated large amounts of cGMP (D).
|E) The quantification of cGMP-positive cells in the ONL and the PDE6A pixel intensity in the OS (arbitrary units; AU) shows an inverse correlation. Images are representative for immunostaining performed on retinal sections from at least three independent animals for each genotype.|
Figure 2. Photoreceptor cell death and survival.
A-C) The TUNEL assay in the wt retina occasionally labeled cells dying due to developmental processes.
D-F) In Pde6a mutants, photoreceptor cell death was dramatically increased.
The images show the situation at P12, P15 and P21, time-points corresponding to the peak of cell death..
G, H) The line graph at the bottom left (G) illustrates the progression of photoreceptor cell death as evidenced by the TUNEL assay in different Pde6a mutants. The green line corresponds to the V685M*R562W mutant.
The peak times as well as the peak amplitudes correspond to the speed of retinal degeneration, which is illustrated by the loss of photoreceptors (H).
Images are representative for TUNEL assays performed on retinal sections from at least three independent animals; quantifications in G, H include data from 3-7 animals per genotype and time-point.
Figure 3. Photoreceptor degeneration in Pde6a mutants correlates with calpain, not caspase, activity.
Calpain activity is often associated with non-apoptotic forms of cell death, and in all Pde6a mutants it is strongly correlated in time with the progression of photoreceptor cell death.
A) Quantification of caspase-3-positive cells, which shows only extremely low numbers of cells at the respective peaks of degeneration, with no significant (n.s.) differences to wt.
B) The progression of calpain activity was analyzed over time and showed a strong correlation to the extent of cell death and the progression of retinal degeneration.
Akbergenov R, Duscha S, Fritz AK, Juskeviciene R, Oishi N, Schmitt K, Shcherbakov D, Teo Y, Boukari H, Freihofer P, Isnard-Petit P, Oettinghaus B, Frank S, Thiam K, Rehrauer H, Westhof E, Schacht J, Eckert A, Wolfer D, Böttger EC.
Mutant MRPS5 affects mitoribosomal accuracy and confers stress-related behavioral alterations.
EMBO Rep. 2018 Sep 20.
Clemen CS, Winter L, Strucksberg KH, Berwanger C, Türk M, Kornblum C, Florin A, Aguilar-Pimentel JA, Amarie OV, Becker L, Garrett L, Hans W, Moreth K, Neff F, Pingen L, Rathkolb B, Rácz I, Rozman J, Treise I, Fuchs H, Gailus-Durner V, de Angelis MH, Vorgerd M, Eichinger L, Schröder R.
The heterozygous R155C VCP mutation: Toxic in humans! Harmless in mice?
Biochem Biophys Res Commun. 2018 Sep 18.
Andrew RJ, Fernandez CG, Stanley M, Jiang H, Nguyen P, Rice RC, Buggia-Prévot V, De Rossi P, Vetrivel KS, Lamb R, Argemi A, Allaert ES, Rathbun EM, Krause SV, Wagner SL6, Parent AT, Holtzman DM, Thinakaran G.
Lack of BACE1 S-palmitoylation reduces amyloid burden and mitigates memory deficits in transgenic mouse models of Alzheimer's disease.
Proc Natl Acad Sci U S A. 2017 Nov 7.
Hu LR, Ackermann MA, Hecker PA, Prosser BL, King B, O'Connell KA, Grogan A, Meyer LC, Berndsen CE, Wright NT, Jonathan Lederer W, Kontrogianni-Konstantopoulos A.
Deregulated Ca2+ cycling underlies the development of arrhythmia and heart disease due to mutant obscurin.
Sci Adv. 2017 Jun 7.
Kauskot A, Poirault-Chassac S, Adam F, Muczynski V, Aymé G, Casari C, Bordet JC, Soukaseum C, Rothschild C, Proulle V, Pietrzyk-Nivau A, Berrou E, Christophe OD, Rosa JP, Lenting PJ, Bryckaert M, Denis CV, Baruch D.
LIM kinase/cofilin dysregulation promotes macrothrombocytopenia in severe von Willebrand disease-type 2B.
JCI Insight. 2016 Oct 6.
Le Stunff C, Tilotta F, Sadoine J, Le Denmat D, Briet C, Motte E, Clauser E, Bougnères P, Chaussain C, Silve C.
Knock-in of the Recurrent R368X Mutation of PRKAR1A that Represses cAMP-Dependent Protein Kinase A Activation: A Model of Type 1 Acrodysostosis.
J Bone Miner Res. 2016 Sep 2.
Jiao K, Sahaboglu A, Zrenner E, Ueffing M, Ekström PA, Paquet-Durand F.
Efficacy of PARP inhibition in Pde6a mutant mouse models for retinitis pigmentosa depends on the quality and composition of individual human mutations.
Cell Death Discov. 2016 Jul 4.
Adam F, Casari C, Prévost N, Kauskot A, Loubière C, Legendre P, Repérant C, Baruch D, Rosa JP, Bryckaert M, de Groot PG, Christophe OD, Lenting PJ, Denis CV.
A genetically-engineered von Willebrand disease type 2B mouse model displays defects in hemostasis and inflammation.
Sci Rep. 2016 May 23.
Qu Y, Misaghi S, Newton K, Maltzman A, Izrael-Tomasevic A, Arnott D, Dixit VM.
NLRP3 recruitment by NLRC4 during Salmonella infection.
J Exp Med. 2016 May 2.
Yavari A, Stocker CJ, Ghaffari S, Wargent ET, Steeples V, Czibik G, Pinter K, Bellahcene M, Woods A, Martínez de Morentin, Cansell C, Lam BY, Chuster A, Petkevicius K, Nguyen-Tu MS, Martinez-Sanchez A, Pullen TJ, Oliver PL, Stockenhuber A, Nguyen C, Lazdam M, O'Dowd JF, Harikumar P, Tóth M, Beall C, Kyriakou T, Parnis J, Sarma D, Katritsis G, Wortmann DD, Harper AR, Brown LA, Willows R, Gandra S, Poncio V, de Oliveira Figueiredo MJ, Qi NR, Peirson SN, McCrimmon RJ, Gereben B, Tretter L, Fekete C, Redwood C, Yeo GS, Heisler LK, Rutter GA, Smith MA, Withers DJ, Carling D, Sternick EB, Arch JR, Cawthorne MA, Watkins H, Ashrafian H.
Chronic Activation of γ2 AMPK Induces Obesity and Reduces β Cell Function.
Cell Metab. 2016 Apr 26.
Ness JK, Skiles AA, Yap EH, Fajardo EJ, Fiser A, Tapinos N.
Nuc-ErbB3 regulates H3K27me3 levels and HMT activity to establish epigenetic repression during peripheral myelination.
Glia. 2016 Mar 28.
Viuff D, Antunes F, Evans L, Cameron J, Dyrnesli H, Thue Ravn B, Stougaard M, Thiam K, Andersen B, Kjærulff S, Howard KA.
Generation of a double transgenic humanized neonatal Fc receptor (FcRn)/albumin mouse to study the pharmacokinetics of albumin-linked drugs.
J Control Release. 2016 Feb 10.
Butcher AJ, Bradley SJ, Prihandoko R, Brooke SM, Mogg A, Bourgognon JM, Macedo-Hatch T, Edwards JM, Bottrill AR, Challiss RA, Broad LM, Felder CC, Tobin AB.
An antibody biosensor establishes the activation of the M1 muscarinic acetylcholine receptor during learning and memory.
J Biol Chem. 2016 Jan 29.
Chevessier F, Schuld J, Orfanos Z, Plank AC, Wolf L, Maerkens A, Unger A, Schlötzer-Schrehardt U, Kley RA, Von Hörsten S, Marcus K, Linke WA, Vorgerd M, van der Ven PF, Fürst DO, Schröder R.
Myofibrillar instability exacerbated by acute exercise in filaminopathy.
Hum Mol Genet. 2015 Dec 20.
Yzaguirre AD, Padmanabhan A, de Groh ED, Engleka KA, Li J, Speck NA, Epstein JA.
Loss of neurofibromin Ras-GAP activity enhances the formation of cardiac blood islands in murine embryos.
Elife. 2015 Oct 13.
Sothilingam V, Garcia Garrido M, Jiao K, Buena-Atienza E, Sahaboglu A, Trifunović D, Balendran S, Koepfli T, Mühlfriedel R, Schön C, Biel M, Heckmann A, Beck SC, Michalakis S, Wissinger B, Seeliger MW, Paquet-Durand F.
Retinitis pigmentosa: impact of different Pde6a point mutations on the disease phenotype.
Hum Mol Genet. 2015 Oct 1.
Mellgren AE, Bruland O, Vedeler A, Saraste J, Schönheit J, Bredrup C, Knappskog PM, Rødahl E.
Development of congenital stromal corneal dystrophy is dependent on export and extracellular deposition of truncated decorin.
Invest Ophthalmol Vis Sci. 2015 May 31.
Smallie T, Ross EA, Ammit AJ, Cunliffe HE, Tang T, Rosner DR, Ridley ML, Buckley CD, Saklatvala J, Dean JL, Clark AR.
Dual-Specificity Phosphatase 1 and Tristetraprolin Cooperate To Regulate Macrophage Responses to Lipopolysaccharide.
J Immunol. 2015 May 27.
Ross EA, Smallie T, Ding Q, O'Neil JD, Cunliffe HE, Tang T, Rosner DR, Klevernic I, Morrice NA, Monaco C, Cunningham AF, Buckley CD, Saklatvala J, Dean JL, Clark AR.
Dominant Suppression of Inflammation via Targeted Mutation of the mRNA Destabilizing Protein Tristetraprolin.
J Immunol. 2015 May 22.
Mandal P, Berger SB, Pillay S, Moriwaki K, Huang C, Guo H, Lich JD, Finger J, Kasparcova V, Votta B, Ouellette M, King BW, Wisnoski D, Lakdawala AS, DeMartino MP, Casillas LN, Haile PA, Sehon CA, Marquis RW, Upton J, Daley-Bauer LP, Roback L, Ramia N, Dovey CM, Carette JE, Chan FK, Bertin J, Gough PJ, Mocarski ES, Kaiser WJ.
RIP3 induces apoptosis independent of pronecrotic kinase activity.
Mol Cell. 2014 Nov 20.
Clemen CS, Stöckigt F, Strucksberg KH, Chevessier F, Winter L, Schütz J, Bauer R, Thorweihe JM, Wenzel D, Schlötzer-Schrehardt U, Rasche V, Krsmanovic P, Katus HA, Rottbauer W, Just S, Müller OJ, Friedrich O, Meyer R, Herrmann H, Schrickel JW, Schröder R
The toxic effect of R350P mutant desmin in striated muscle of man and mouse.
Acta Neuropathol. 2014 Nov 14.
Fantin A, Herzog B, Mahmoud M, Yamaji M, Plein A, Denti L, Ruhrberg C, Zachary I.
Neuropilin 1 (NRP1) hypomorphism combined with defective VEGF-A binding reveals novel roles for NRP1 in developmental and pathological angiogenesis.
Development. 2014 Jan 8.
Puszyk W, Down T, Grimwade D, Chomienne C, Oakey RJ, Solomon E, Guidez F.
The epigenetic regulator PLZF represses L1 retrotransposition in germ and progenitor cells.
EMBO J. 2013 May 31.
Chen CM, Bentham J, Cosgrove C, Braganca J, Cuenda A, Bamforth SD, Schneider JE, Watkins H, Keavney B, Davies B, Bhattacharya S.
Functional Significance of SRJ Domain Mutations in CITED2.
PLoS One. 2012 Oct 17.
Kollmann K, Damme M, Markmann S, Morelle W, Schweizer M, Hermans-Borgmeyer I, Röchert AK, Pohl S, Lübke T, Michalski JC, Käkelä R, Walkley SU, Braulke T.
Lysosomal dysfunction causes neurodegeneration in mucolipidosis II 'knock-in' mice.
Brain. 2012 Sep.
Nogales-Gadea G, Pinós T, Lucia A, Arenas J, Camara Y, Brull A, de Luna N, Martín MA, Garcia-Arumí E, Martí R, Andreu AL.
Knock-in mice for the R50X mutation in the PYGM gene present with McArdle disease.
Brain. 2012 Jul.
Drost R, Bouwman P, Rottenberg S, Boon U, Schut E, Klarenbeek S, Klijn C, van der Heijden I, van der Gulden H, Wientjens E, Pieterse M, Catteau A, Green P, Solomon E, Morris JR, Jonkers J.
BRCA1 RING function is essential for tumor suppression but dispensable for therapy resistance.
Cancer Cell. 2011 Dec 13.
Shu X, Luhmann UF, Aleman TS, Barker SE, Lennon A, Tulloch B, Chen M, Xu H, Jacobson SG, Ali R, Wright AF.
Characterisation of a C1qtnf5 Ser163Arg Knock-In Mouse Model of Late-Onset Retinal Macular Degeneration.
PLoS One. 2011 Nov 16.
Daroszewska A, van 't Hof RJ, Rojas JA, Layfield R, Landao-Basonga E, Rose L, Rose K, Ralston SH.
A point mutation in the ubiquitin-associated domain of SQSMT1 is sufficient to cause a Paget's disease-like disorder in mice.
Hum Mol Genet. 2011 Jul 15.
Ferry G, Giganti A, Coge F, Bertaux F, Thiam K, Boutin JA.
Functional invalidation of the autotaxin gene by a single amino acid mutation in mouse is lethal.
FEBS Lett. 2007 Jul 24.
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