David L. Nelson, Ph.D.
Principal Investigator
Baylor College of Medicine
Houston

Through funding by FRAXA Research Foundation, a Fragile X Mutant Mouse facility has been established at Baylor College of Medicine. A list of mouse strains is provided below. As the facility gets up to speed, it is anticipated that in addition to breeding pairs, animals in numbers sufficient for experimentation as well as tissues will be provided, reducing the need for others to establish their own colonies. These latter services may require small fees if demand becomes significant. Requesting groups will be expected to reimburse for shipping charges and for some strains, there will be a Materials Transfer Agreement with the BCM Licensing group. Please contact David Nelson directly at nelson@bcm.edu to request mice or with any questions or concerns.

Available animals:

Fmr1 KO This is the original Fmr1 knockout animal that carries an insertion in exon 5 (Bakker et al., 1994). It is a protein null, although Fmr1 mRNA is still present (Yan et al., 2004). Mice available from the BCM facility have been backcrossed over 20 generations to the C57/Bl6 strain.

Fmr1 KO2 This is a new null allele at Fmr1 generated by deletion of the promoter and first exon of Fmr1 (Mientjes et al., 2006). It is both protein and mRNA null. This mutation is the same as is produced by Cre-mediated excision of the loxP sites present in the Fmr1 cKO described below.

Fmr1 cKO (conditional knockout) This mouse carries loxP sites that were introduced by homologous recombination to flank the promoter and first exon of Fmr1 (Mientjes et al., 2006). Cre-mediated recombination between the loxP sites will eliminate Fmr1 expression, producing a KO2 deletion (Koekkoek et al., 2005).

Fmr1 cON (conditional restoration) This strain carries a neomycin selection cassette that is flanked by loxP sites in the first intron of Fmr1(Mientjes et al., 2006). Due to the presence of an oppositely oriented transcribed gene in intron 1, expression of Fmr1 is greatly reduced. We estimate less than 10% of normal levels of FMRP are present in brains of these animals. Cre-mediated recombination between the loxP sites eliminates the interfering neomycin gene and restores normal expression levels of Fmr1.

Fmr1 YAC transgenic mice (TG298) These transgenic animals carry a yeast artificial chromosome that expresses human FMR1 (Peier et al., 2000; Spencer et al., 2008). Total FMRP levels are elevated several fold over wildtype mice, providing an animal model that overexpresses FMRP with all alternatively spliced isoforms. These animals were created and maintained on the C57/Bl6 strain.

Fmr1 CGG expansion These animals carry an expanded (~100 triplets) CGG repeat in the mouse Fmr1 gene. They were created by the Oostra group to serve as a model for repeat instability and expansion, with the expectation that they would accurately model the common mutation in Fragile X syndrome (Bontekoe et al., 2001). Unfortunately, the repeat was not found to be unstable at the levels necessary to model the disease, and larger repeats have not demonstrated the methylation that is found in humans. However, this line has become a very important model for FXTAS, the late age of onset neurodegenerative disorder seen in approximately 50% of males carrying premutations (Willemsen, 2003). The animals currently available at BCM carry ~150 repeats, and have been backcrossed into C57/Bl6 for more than ten generations.

FMR1 CGG90YAC transgenics Additional lines carrying a human FMR1 gene with expanded CGG repeats as a YAC transgene were developed by the Nelson group for study of CGG repeat instability. These are also models for FXTAS.

Fmr1 Paralogs

In mammals, there are two proteins that are highly identical to Fmrp, known as Fxr1p and Fxr2p. These carry the same functional domains as Fmr1, bind RNA, can be found in complexes with Fmrp, and are likely to function with Fmrp in some of its roles. Understanding the functions of these genes has assisted with determining the role of Fmrp and the consequences of its absence. For example, animals lacking both Fmrp and Fxr2p exhibit more profound phenotypes than those lacking either alone. Knockout and conditional alleles for these genes have been produced and are proposed to be offered by the resource. These are described below.

Fxr2 KO Fxr2 is an autosomal paralog of Fmr1. The Oostra and Nelson groups developed a knockout mouse for this gene by insertion of a selection cassette into exon 7, eliminating protein expression (Bontekoe et al., 2002). These animals have been backcrossed into C57/Bl6 for more than 20 generations.

Fxr1 KO Fxr1 is an autosomal paralog of Fmr1. The Oostra and Nelson groups have developed a series of alleles at Fxr1. The KO allele is a deletion of the promoter and first exon, and is protein and RNA null (Mientjes et al., 2004). Homozygotes are born alive but die shortly after birth due to muscle abnormalities. These animals are maintained as heterozygotes, and have been backcrossed into C57/Bl6 for more than 20 generations. The deletion present in these KO animals is the same as that created by Cre mediated deletion of the cKO allele described below.

Fxr1 cKO (conditional knockout) This mouse carries loxP sites that were introduced by homologous recombination to flank the promoter and first exon of Fxr1. Cre-mediated recombination between the loxP sites will eliminate Fxr1 expression, producing an Fxr1 KO deletion. These animals are viable and fertile and have been maintained on a C57/Bl6 background for more than 10 generations.

Fxr1 cON (conditional restoration) This strain carries a neomycin selection cassette that is flanked by loxP sites in the first intron of Fxr1. Due to the presence of an oppositely oriented transcribed gene in intron 1, expression of Fxr1 is greatly reduced, resulting in a hypomorphic mutation. We estimate less than 10% of normal levels of FMRP are present in tissues of these animals. As a result, homozygous animals are not healthy, and only survive for 3-6 months. They are not fertile. Cre-mediated recombination between the loxP sites eliminates the interfering neomycin gene and restores normal expression levels of Fxr1. These animals are maintained as heterozygotes on a C57/Bl6 background. They have been backcrossed more than 10 generations.

Fmr1/Fxr2 Double knockout and Fmr1KO/Fxr2+/- heterozygotes The Nelson group has made use of combinations of Fmr1 and Fxr2 mutations to elicit additional phenotypes such as hyperactivity and circadian rhythm anomalies (Spencer et al., 2006; Zhang et al., 2008). The double KO animals are difficult to maintain as a line, and need to be produced from a double heterozygote cross. We have provided these animals to other investigators; we know that they travel well.

Cre expression lines Many transgenic animals have been developed that express Cre recombinase in specific tissues or upon induction. The Nelson group has imported several of these, and has demonstrated Purkinje cell specific ablation of Fmr1 and Fxr1 for phenotypic studies. Many other Cre expression lines are available locally at BCM. Subject to the agreement by the original developers of these lines, we could offer Fmr1 and Fxr1 conditional alleles crossed into these Cre expression animals.

Additional strains can be made available upon request. Please inquire. The FRAXA web site will be updated with information regularly.

References

Bakker CE, Verheij C, Willemsen R, Vanderhelm R, Oerlemans F, Vermey M, Bygrave A, Hoogeveen AT, Oostra BA, Reyniers E, Deboulle K, Dhooge R, Cras P, Van Velzen D, Nagels G, Martin JJ, Dedeyn PP, Darby JK, Willems PJ (1994) Fmr1 knockout mice: A model to study fragile X mental retardation. Cell 78:23-33.

Bontekoe CJ, Bakker CE, Nieuwenhuizen IM, van Der Linde H, Lans H, de Lange D, Hirst MC, Oostra BA (2001) Instability of a (CGG)(98) repeat in the Fmr1 promoter. Hum Mol Genet 10:1693-1699.

Bontekoe CJ, McIlwain KL, Nieuwenhuizen IM, Yuva-Paylor LA, Nellis A, Willemsen R, Fang Z, Kirkpatrick L, Bakker CE, McAninch R, Cheng NC, Merriweather M, Hoogeveen AT, Nelson D, Paylor R, Oostra BA (2002) Knockout mouse model for Fxr2: a model for mental retardation. Hum Mol Genet 11:487-498.

Koekkoek SK, Yamaguchi K, Milojkovic BA, Dortland BR, Ruigrok TJ, Maex R, De Graaf W, Smit AE, VanderWerf F, Bakker CE, Willemsen R, Ikeda T, Kakizawa S, Onodera K, Nelson DL, Mientjes E, Joosten M, De Schutter E, Oostra BA, Ito M, De Zeeuw CI (2005) Deletion of FMR1 in Purkinje cells enhances parallel fiber LTD, enlarges spines, and attenuates cerebellar eyelid conditioning in Fragile X syndrome. Neuron 47:339-352.

Mientjes EJ, Nieuwenhuizen I, Kirkpatrick L, Zu T, Hoogeveen-Westerveld M, Severijnen L, Rifé M, Willemsen R, Nelson DL, Oostra BA (2006) The generation of a conditional Fmr1 knock out mouse model to study Fmrp function in vivo. Neurobiology of Disease 21:549-555.

Mientjes EJ, Willemsen R, Kirkpatrick LL, Nieuwenhuizen IM, Hoogeveen-Westerveld M, Verweij M, Reis S, Bardoni B, Hoogeveen AT, Oostra BA, Nelson DL (2004) Fxr1 knockout mice show a striated muscle phenotype: implications for Fxr1p function in vivo. Hum Mol Genet 13:1291-1302.

Peier AM, McIlwain KL, Kenneson A, Warren ST, Paylor R, Nelson DL (2000) (Over)correction of FMR1 deficiency with YAC transgenics: behavioral and physical features. Hum Mol Genet 9:1145-1159.

Spencer CM, Graham DF, Yuva-Paylor LA, Nelson DL, Paylor R (2008) Social behavior in Fmr1 knockout mice carrying a human FMR1 transgene. Behav Neurosci 122:710-715.

Spencer CM, Serysheva E, Yuva-Paylor LA, Oostra BA, Nelson DL, Paylor R (2006) Exaggerated behavioral phenotypes in Fmr1/Fxr2 double knockout mice reveal a functional genetic interaction between Fragile X-related proteins. Hum Mol Genet 15:1984-1994.

Willemsen R, Hoogeveen-Westerveld, M., Reis, S., Holstege, J., Severijnen, L., Nieuwenhuizen, I., Schrier, M., VanUnen, L., Tassone, F., Hoogeveen, A., Hagerman, P., Mientjes, E., Oostra, B. (2003) The FMR1 CGG repeat mouse displays ubiquitin-positive intranuclear neuronal inclusions; implications for the cerebellar tremor/ataxia syndrome. Human Molecular Genetics 12:949-959.

Yan QJ, Asafo-Adjei PK, Arnold HM, Brown RE, Bauchwitz RP (2004) A phenotypic and molecular characterization of the fmr1-tm1Cgr fragile X mouse. Genes Brain Behav 3:337-359.

Zhang J, Fang Z, Jud C, Vansteensel MJ, Kaasik K, Lee CC, Albrecht U, Tamanini F, Meijer JH, Oostra BA, Nelson DL (2008) Fragile x-related proteins regulate Mammalian circadian behavioral rhythms. Am J Hum Genet 83:43-52.