FRAXA-DVI - facility to test compounds in Fragile X mice
FRAXA funds a Drug Validation Initiative in Chile run by Dr. Patricia Cogram. At this facility we can run a series of behavioral and anatomical tests of potential therapeutic compounds using FMR1 knockout mice, at very low or no cost. Please see FRAXA-DVI or Dr. Michael Tranfaglia at email@example.com for more information.
Mutant Mouse Models – FMR1 and Paralogs
A Fragile X Mutant Mouse facility was established in 2009 at Baylor College of Medicine. This facility is funded by FRAXA with support from The Meadows Foundation. The BCM facility’s goal is to provide convenient and rapid access to new mouse models to investigators worldwide on request for modest costs of shipping. More than a dozen advanced mouse strains are available to researchers; the laboratory of David L. Nelson, PhD, is accepting requests.
Mice are typically provided without restrictions (other than those required by the BCM standard Materials Transfer Agreement, which limits commercial use and re-distribution). In a small number of cases, where specific tissues and/or large numbers of animals have been requested, the Nelson group has engaged in a collaborative arrangement to acknowledge the extra time and effort required by Nelson lab personnel. Please contact David Nelson directly at firstname.lastname@example.org to request mice or with any questions or concerns.
Mutant Mouse Strains Available
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.
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.
Knockout Mice are also available from Jackson Laboratory.
FMR1 Knockout Rats
This model contains a deletion of the Fragile X mental retardation 1 gene (Fmr1). Mutations in Fmr1 result in Fragile X syndrome, the leading monogenic cause of autism, making this rat useful for the study of both Fragile X syndrome and autism.
Sold by Horizon Discovery Group. Visit Horizon Discovery.
Human Fragile X Cortical Neural Stem Cells
Human cortical neural stem cells that carry the Fragile X mutation are available for distribution to interested researchers. These cells are grown as neurospheres and are mainly neural progenitor cells. They can be differentiated into neurons and astrocytes that lack FMRP with long-term culturing. For more information about these cells please refer to Svendsen et al., J Neuroscience Methods 85:141-163 (1998), and contact: Dr. Anita Bhattacharyya at the University of Wisconsin-Madison, Waisman Center www.waisman.wisc.edu/faculty/bhattacharyya.html
Antibodies to the Fragile X Protein
Several antibodies to the FMRP are available at nominal cost from the DHSB at the University of Iowa. Several antibodies to mouse and drosophila fmrp are available.For details, visit: http://dshb.biology.uiowa.edu/
Human Brain Tissue
FRAXA’s primary mission is to speed up progress towards effective treatments and a cure for Fragile X. As “bottlenecks” are identified we will try to facilitate solutions, so please contact us for additional scientific resource requests.