X-ray v2 06/AUG/2021 RIKENMPP_010_002 test 1 1 testSt test modification

For a long time, fluoroscopic photography with X-ray has been used for noninvasive analyses. Unlike computed tomography (CT) and magnetic resonance imaging (MRI), this technique does not provide stereographic images and only yields planar images of shades. However, it has advantages, such as its relatively simple operation and instrument structure and its ability to provide results in a short time.

These procedures are applicable to the mouse phenotyping platform at the RIKEN BRC. The tests should be performed by trained staff members who understand the safety and hygiene rules required by RIKEN. Any queries, comments, or suggestions relating either to this SOP in general or to a specific problem encountered during a procedure should be addressed to the Morphological Unit Leader. Any deviation from this protocol must be reported to the Morphological Unit Leader.

Testing is carried out in accordance with common animal handling procedures. Coveralls (in the rearing facility) or laboratory coats (in the laboratory facility) must be worn with gloves at all times in the work area. If animals are dropped on the floor, treat them in accordance with the safety and hygiene guidelines from the rearing facility at RIKEN. If something unexpected happens during the primary screening, it must be quickly reported to the Morphological Unit Leader and recorded.

Image: Position of mouse on the X-ray plate for dorso-ventral and lateral orientations

In the ninth chapter "Procedure," "Standard" means phenotypes that are detected in more than 20% of the control mice. Therefore, it is important to recall phenotypes of these mice before analyzing the experimental mice. In each test item, artificial abnormalities should be excluded from the evaluation. X-ray template and posture of the mouse To prevent hair loss by sticky tape, make the tape less cohesive, then put their trunk at the center of the plate (not on the tapes) and fix their limbs and tail using tape. The protocol described herein employs anesthesia using intraperitoneal (IP) administration.

On each experimental day, calibrate the device before image capturing.

X-ray device MX-20-DC12 (Faxitron X-Ray Corporation, USA) X-ray permeable template sheets (Dorso-ventral and lateral orientation) Laminated paper with double-sided tape. Prepare two sheets per trial. For the lateral orientation, set two loops with the tape (4.1 image). Balance

Fine forceps with curved point The forceps are used to pose the mouse on the plate. Anesthetic solution Gauge needle Syringe

Anesthetize the mice. Monitor the mouse carefully until unconsciousness by ensuring the mouse is adequately sedated. Mount the anesthetized mouse in a prone position onto the X-ray plate for the dorso-ventral view. Curve the tail along the left side of the mouse. (Note: this will enable identification of the X-ray as a dorsal or ventral image). Faxitron X-ray system Insert the plate into the Faxitron X-ray system. Set the machine to a voltage of 26 kV, with an exposure time of 10 s. Take an image using the Faxitron DX software. Mount the anesthetized mouse sideways onto another X-ray plate for the lateral orientation view. Place the left limbs on the loops of the tape and the right limbs on the double-sided tape. Curve the tail to the dorsal side. (Note: this will enable identification of the X-ray as a lateral image). Repeat steps 9.4.1 - 9.4.3. Place the mouse back in its home cage. Analyze the images. Record the grade, number, and/or comments for the following 61 items related to bone characteristics. Comment Head Skull shape 0=Standard 1=Domed 2=Osteoma 3=Nasal deformity 4=Overall deformation Other Zygomatic arch 0=Standard 1=Thin 2=Thick 3=Depressed 4=Enlarged Other Maxilla/Pre-maxilla 0=Standard 1=Enlarged 2=Osteoma 3=Overall deformation Other Mandibles 0=Standard 1=Osteoma 2=Odd angle 3=Overall deformation Other Teeth 0=Standard 1=Short 2=Long 3=Misaligned 4=Few or absent Other Trunk Scapulae 0=Standard 1=Hole Other Clavicle 0=Standard 1=Absent 2=Hypoplasia 3=Deformed acromial extremity Other Number of ribs right Number of ribs left Shape of ribs Assess malformations of the ribs, except for fusions and/or transitional vertebrae. 0=Standard 1=Thin 2=Thick 3=Truncated 4=Long 5=Bowed 6=Cervical rib Other Fusion of ribs 0=Standard 1=Fused Shape of ribcage 0=Standard 1=Caved-in 2=Enlarged 3=Small Other Missing cranial rib 0=No 1=Yes Pelvis 0=Standard 1=Enlarged 2=Small 3=Abnormal ilium 4=Abnormal ischium 5=Abnormal pubis 6=Deformed iliopectineal eminence 7=Overall deformation Other Spine Shape of spine 0=Standard 1=Abnormal curvature 2=Thin 3=Thick Other Scoliosis 0=No 1=Yes Kyphosis 0=No 1=Yes Lordosis 0=No 1=Yes Transitional vertebrae 0=No 1=Yes Number of cervical vertebrae Number of thoracic vertebrae Number of lumbar vertebrae Number of pelvic vertebrae Number of caudal vertebrae The number of caudal vertebrae of the mice with cut tail is omitted from the assessment. Shape of vertebrae Assess malformations (e.g., fusion) of the vertebral bodies and the vertebral arches, except for transitional vertebrae. (There are 5 regions: cervical, thoracic, lumbar, sacral and caudal.) 0=Standard 1=Deformation on one region of vertebrae 2=Deformation on regions of vertebrae Other Shape of cervical vertebrae Assess malformations of the cervical vertebra, except for fusions and/or transitional vertebrae. 0=Standard 1=Enlarged 2=Small 3=Hemivertebrae 4=Deformed ventral tubercle of atlas 5=Split Other Shape of thoracic vertebrae Assess malformations of the thoracic vertebra, except for fusions and/or transitional vertebrae. 0=Standard 1=Enlarged 2=Small 3=Hemivertebrae Other Shape of lumbar vertebrae Assess malformations of the lumbar vertebra, except for fusions and/or transitional vertebrae. 0=Standard 1=Enlarged 2=Small 3=Hemivertebrae Other Shape of sacral vertebrae Assess malformations of the sacral vertebra, except for fusions and/or transitional vertebrae. 0=Standard 1=Enlarged 2=Small 3=Hemivertebrae Other Shape of caudal vertebrae Assess malformations of the caudal vertebra, except for fusions and/or transitional vertebrae. 0=Standard 1=Enlarged 2=Small 3=Hemivertebrae Other Fusion of cervical vertebrae Assess fusions of the cervical vertebra, except for transitional vertebrae. 0=Standard 1=Fused at a joint 2=Fused at joints Other Fusion of thoracic vertebrae Assess fusions of the thoracic vertebra, except for transitional vertebrae. 0=Standard 1=Fused at a joint 2=Fused at joints Other Fusion of lumbar vertebrae Assess fusions of the lumbar vertebra, except for transitional vertebrae. 0=Standard 1=Fused at a joint 2=Fused at joints Other Fusion of sacral vertebrae Assess fusions of the sacral vertebra, except for transitional vertebrae. 0=Standard 1=Unfused 2=Extra fused Other Fusion of caudal vertebrae Assess fusions of the caudal vertebra, except for transitional vertebrae. 0=Standard 1=Fused at a joint 2=Fused at joints Other Processes on vertebrae Assess malformations (e.g., fusion) of the vertebral processes, except for transitional vertebrae. (There are 5 regions: cervical, thoracic, lumbar, sacral and caudal.) 0=Standard 1=Deformation on one region of vertebrae 2=Deformation on regions of vertebrae Other Cervical processes Assess malformations of the vertebral processes on the cervical vertebrae, except for fusions and/or transitional vertebrae. 0=Standard 1=Truncated 2=Enlarged 3=Bowed 4=Deformed spinous process on Ce2 Other Thoracic processes Assess malformations of the vertebral processes on the thoracic vertebrae, except for fusions and/or transitional vertebrae. 0=Standard 1=Truncated 2=Enlarged 3=Bowed 4=Long spinous process on T1 and/or short spinous process on T2 5=Deformed spinous process on T10 6=Various deformations Other Lumbar processes Assess malformations of the vertebral processes on the lumbar vertebrae, except for fusions and/or transitional vertebrae. 0=Standard 1=Truncated 2=Enlarged 3=Bowed Other Sacral processes Assess malformations of the vertebral processes on the sacral vertebrae, except for fusions and/or transitional vertebrae. 0=Standard 1=Truncated 2=Enlarged 3=Abnormal sacral foramina Other Caudal processes Assess malformations of the vertebral processes on the caudal vertebrae, except for fusions and/or transitional vertebrae. 0=Standard 1=Truncated 2=Enlarged Other Fusion of cervical processes Assess fusions of the vertebral processes on the cervical vertebrae, except for transitional vertebrae. 0=Standard 1=Fused between a pair 2=Fused between pairs Other Fusion of thoracic processes Assess fusions of the vertebral processes on the thoracic vertebrae, except for transitional vertebrae. 0=Standard 1=Fused between a pair 2=Fused between pairs Other Fusion of lumbar processes Assess fusions of the vertebral processes on the lumbar vertebrae, except for transitional vertebrae. 0=Standard 1=Fused between a pair 2=Fused between pairs Other Fusion of sacral processes Assess fusions of the vertebral processes on the sacral vertebrae, except for transitional vertebrae. 0=Standard 1=Unfused 2=Extra fused Other Fusion of caudal processes Assess fusions of the vertebral processes on the caudal vertebrae, except for transitional vertebrae. 0=Standard 1=Fused between a pair 2=Fused between pairs Other Limb Humerus 0=Standard 1=Thin 2=Thick 3=Short 4=Long 5=Bowed 6=Thick or thin cortical bone 7=Overall deformation Other Radius 0=Standard 1=Thin 2=Thick 3=Short 4=Long 5=Bowed 6=Fused with ulna 7=Thick or thin cortical bone 8=Overall deformation Other Ulna 0=Standard 1=Thin 2=Thick 3=Short 4=Long 5=Bowed 6=Fused with radius 7=Thick or thin cortical bone 8=Overall deformation Other Femur 0=Standard 1=Thin 2=Thick 3=Short 4=Long 5=Bowed 6=Thick or thin cortical bone 7=Overall deformation Other Tibia 0=Standard 1=Thin 2=Thick 3=Short 4=Long 5=Bowed 6=Fused with fibula 7=Thick or thin cortical bone 8=Overall deformation Other Tibia length (mm) Measure the length of the right tibia (the distance from the proximal end to the distal end) on the millimeter length scale. Fibula 0=Standard 1=Thin 2=Thick 3=Short 4=Long 5=Bowed 6=Fused with tibia Other Digits 0=Standard 1=Thin 2=Thick 3=Deformed 4=Syndactyly 5=Brachydactyly 6=Polydactyly 7=Polysyndactyly 8=Various deformations Other Number of digits Syndactyly 0=Standard 1=Of only forelimb 2=Of only hindlimb 3=Of forelimb and hindlimb Other Brachydactyly 0=Standard 1=Of only forelimb 2=Of only hindlimb 3=Of forelimb and hindlimb Other Polydactyly 0=Standard 1=Of only forelimb 2=Of only hindlimb 3=Of forelimb and hindlimb Other Joints Assess malformations of the joints, except for vertebral fusion. 0=Standard 1=Unfused 2=Extra fused 3=Luxation or Subluxation 4=Deformed Other Alive Alive Dead

[2021.4] For v2.10, options were added: options 3-4 in "Skull shape;" option 3 in "Maxilla/Pre-maxilla;" option 3 in "Mandibles;" option 3 in "Clavicle;" options 6-7 in "Pelvis;" options 1-2 in "Shape of vertebrae;" options 1-2 in "Processes on vertebrae;" option 4 in "Cervical processes;" options 4-6 in "Thoracic processes;" options 6-7 in "Humerus;" options 7-8 in "Radius;" options 7-8 in "Ulna;" options 6-7 in "Femur;" options 7-8 in "Tibia;" options 1-8 in "Digits." [2018.11] A new version of the X-ray method (Ver. 2.00) was added.