DYSFERLIN BACKGROUND
Research Tips
DYSFERLIN BACKGROUND
Research Tips
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Research Tips
Below is a list of protocols, procedures, and advice that the Jain Foundation has compiled to help researchers engaged in dysferlin research. This list focuses on areas that are specific or unique to dysferlin research or that we are often asked.
Please be mindful that the field of dysferlinopathy is very young with many uncertainties and assumptions. Therefore, we encourage all researchers to keep an open mind when approaching their dysferlin research and to consider the goal of their research when assessing whether these tips are applicable.
If there is something that you need that isn’t listed or that you would recommend that we add to the list, please contact us at admin@jain-foundation.org. We are here to help and welcome your input.
Our experience with the different dysferlin deficient mouse models has resulted in a preference for the BlaJ strain developed by Dr. Isabelle Richard at Genethon (PMID: 20154340) for most applications that don’t require a specific type of DYSF mutation. The genetic dysferlin mutation in the BlaJ line is an ETn retrotransposon (5-6kb) inserted in intron 4 of the dysferlin gene, which results in no detectable dysferlin expressed by these animals.
In the BlaJ strain (Jax strain #012767), the progressive muscular dystrophy (prmd) allele from the A/J inbred strain is crossed onto the C57BL/6 genetic background. Therefore, The C57BL/6 (Jax strain# 000664) strain can be used as a wildtype control.
Dr. Richard donated the strain to The Jackson Laboratory in 2010. Please note that if you search for the BlaJ strain in the regular JAX catalog it will say it is cryopreserved. However, the Jain Foundation has a live colony of the BlaJ mice that we supply to researchers worldwide free of charge, including aged mice. To request BlaJ from the Jain Foundation colony mice send an email to the Jain Foundation (admin@jain-foundation.org) indicating the number, sex, and age for the mice you desire. Use of this line should acknowledge Dr. Isabelle Richard for her development, characterization, and donation of this line to the public repository.
There are a growing number man-made and naturally occurring dysferlin deficient mouse lines, including those with DYSF point mutations which can be used for genetic editing and nonsense readthrough evaluations. For a full list of available animals models, please visit our research tools page on the topic.
Dysferlinopathy has a pronounced bias toward affecting certain muscles. While we don’t yet understand why this is the case, it is important to take into consideration when evaluating mice, as many of the traditionally studied lower limb muscles are not very affected in dysferlin null mice. It is also important to note that histological features such as centrally located nuclei, necrosis, and fat and immune cell infiltration are not readily visible in dysferlin deficient mice until after 6 months of age even in the most affected muscles.
The most affected muscles tend to be around the shoulders, hips and spine such as the psoas, gluteus, deltoid, triceps and paraspinal muscles. The more distal one goes, the less affected the muscles tend to be with the tibialis anterior, and gastrocnemius showing very little sign of pathology and seldom any functional weakness. However, all muscles decline with age making the timing of experiments and muscle choice critical to the success of experiments. The mouse diaphragm does show pathology but isn’t a muscle that is routinely studied given the lack of respiratory issues in humans. While dysferlin is highly expressed in the heart, no significant pathology has been noted in either mice or human.
While the TA shows very little pathology even at very late stages of the disease and we don’t recommend using it for histology or functional analysis to assess effectiveness of a treatment, the TA has been used to show differences following injury protocols (PMID: 25920768, 31160583).
This pattern of muscle involvement in mice is markedly different from humans, where the calf muscles are typically one of the earliest and most affected muscle groups.
The psoas muscle is one of the most highly affected muscles in dysferlin deficient mice. However, it can be difficult to dissect, especially as it isn’t a muscle that is routinely used in muscular dystrophy research. If you need support with how to remove the psoas, please contact the Jain Foundation at admin@jain-foundation.org.
Dysferlin is a single pass transmembrane protein that is typically shown just inside the sarcolemma of muscle fibers in muscle cross sections. However, data has shown that most of the dysferlin in a muscle fiber is in the interior of the muscle fiber with much of it being in the muscle t-tubule network. Longitudinal muscle sections and antigen retrieval methods are helpful to visualize dysferlin expression in the t-tubules (PMID: 24302765).
Functional assessments in dysferlin deficient mice rarely show any significant deficit compared to wildtype controls even at advanced ages. This is true of most of the assessments typically used in muscular dystrophy research, including grip tests, downhill treadmill running, and rotor rod. Therefore, we don’t recommend using these functional assessments on dysferlin deficient mice. Instead, we recommend voluntary activity monitoring (especially rearing and distance traveled) and the balance beam assay (see description in next section) as they have shown significant differences compared to wildtype mice , especially with older mice around 12 months of age. We believe that these assessments are useful because they emphasize the use of the hip muscles which are highly affected in mice. You can find information on activity monitoring in dysferlin deficient mice in the following publications (PMID: 28320887, 28334824) and a protocol for the balance beam assay in the next tab.
The balance beam test measures the ability of a mouse to traverse a narrow beam between two raised platforms. The advantage of the balance beam is in the detection of subtle deficits in motor skills and balance that may not be detected by other motor tests, such as the treadmill or rotarod as well as the emphasis on a narrow gait, which counters the compensation mice use for their hip weakness which is to adopt wider stance. Mice with dysferlinopathy have trouble traversing the balance beam and have measurably slower crossing times than wild type mice. Dysferlin deficient mice struggle to place their hind feet on the beam and may occasionally slip off the beam or drag their torso along the beam.
See the following guidelines for the assessment of motor balance and coordination in mice using the Balance beam to determine the specific parameters to use (i.e. width of beam, age, training, etc.)
- Specific force – Multiple researchers have shown no deficits in specific force in limb muscles of dysferlin deficient mice (PMID: 22132688, 20544921, 36613515, 30970035), but specific force deficits have been reported in the diaphragm (PMID: 25815352, 20544921).
- Contraction induced injury – Dysferlin deficient muscle appears highly resistant to lengthening-contraction-induced injury (PMID: 21060153, 19286669), but does show a delayed recovery from injury caused by lengthening contractions (PMID 18815587, 19923419)
- Membrane repair assay – Many researchers have used this assay to assess the functionality of dysferlin. This assay works best when done on teased fibers from dysferlin deficient mice muscles (typically FDB or EDL – PMID: 28750735, 20154340, 35028538). And shows deficits across ages, including in fibers from young mice (i.e. 3 months). The assay is more difficult in cultured dysferlin deficient myotubes, as the heterogeneity of cellular structures can influence injury, dye entry and resealing leading to high variability in outcomes from cell to cell. Still some researchers have persevered to show differences between dysferlin deficient and normal myotubes (PMID: 32881965, 22020321). Membrane repair assays are largely ineffective on mononuclear cells regardless of their dysferlin status. Importantly, correction of membrane repair has been demonstrated to be insufficient to stop the disease in mouse models, suggesting that changes in membrane resealing are not directly linked to the disease process in dysferlin deficiency (PMID: 22666411).
Dysferlinopathy is a late onset slowly progressing disease in both humans and mice. Therefore, functional and histological differences are generally not detectable in dysferlin deficient animals in first 6months, which is profoundly different than the commonly studied muscular dystrophy mouse models such as the mdx. Therefore, we recommend starting histological and functional assessments no earlier than 9 months of age, with 12 – 15-month-old animals showing the largest differences from WT. For most assessments we don’t recommend using animals over 15 months of age as age related muscle changes begin to occur in both the dysferlin deficient and wildtype mice which can confound the evaluations.