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Intrathoracic injection of Adeno‐associated virus serotype‐1 (AAV1) is transported to the spinal cord
Author(s) -
DeRuisseau Lara Roberts,
Sakai Yoshihisa,
Doperalski Nicholas J.,
Fuller David D.,
Byrne Barry J.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a408-b
Subject(s) - adeno associated virus , spinal cord , virology , medicine , virus , serotype , anesthesia , biology , biochemistry , psychiatry , gene , vector (molecular biology) , recombinant dna
Our goal is to develop a clinically relevant method for transgene delivery to both the diaphragm and phrenic motoneuron pool (located in cervical spinal segments C 3 ‐C 5 ). An AAV1 vector containing the ubiquitous cytomegalovirus immediate early gene promoter and the control gene LacZ (AAV1‐CMV‐LacZ) was diluted in sterile saline and mixed with a gel solution to obtain a total volume of 100μ L (4.0 x 10 11 particles). Mice were anesthetized, placed supine, and a 26G needle was inserted between the ribs to a depth of 3 mm. Following the 100μ L injection, mice were held upright for 10 minutes to allow the gel media (containing AAV1‐CMV‐LacZ) to stay in contact with the diaphragm. Four months post‐injection, diaphragm and spinal segments C 3 ‐C 5 were harvested. PCR was performed on isolated DNA using primers specific to the LacZ gene. Mice injected with AAV1‐CMV‐LacZ were positive for LacZ compared to negative controls in both tissues. The protein product of LacZ (beta‐galactosidase; rlu/μ g protein above sham injected control tissue) was present in both the diaphragm (18914 ± 9346) and cervical spinal segments C 3 ‐C 5 (2380 ± 792). We conclude that AAV1‐CMV‐LacZ is transported to the spinal cord after an intrathoracic injection. Ongoing experiments are aimed at co‐localizing AAV1 with retrogradely labled phrenic motoneurons. NIH DK58327 LRD supported by: T32 HD043730/UF Alumni Assoc. Fellowship