Immortalized chromaffin cells disimmortalized with Cre/lox site-directed recombination for use in cell therapy for pain after partial nerve injury.
To prepare immortalized adrenal chromaffin cells for eventual clinical use, the immortalizing oncogene must be removed. We have utilized a Cre-mediated excision of a loxP-flanked Tag sequence to test whether immortalized chromaffin cells could be disimmortalized by this method. Cultures of embryonic rat adrenal cells were immortalized with the tsA-TN retroviral vector encoding the loxP-flanked temperature-sensitive allele of SV40 large T antigen (tsA-TN) and a positive/negative neo/HSV-TK sequence for selection with either G418 or gancyclovir, respectively. These cells were then infected with the 1710-CrePR1 bicistronic retroviral vector coding for a form of Cre modulatable by the synthetic steroid RU486. These immortalized loxTsTag/CrePR1/RAD cells expressed immunoreactivities (ir) for all the catecholamine enzymes: tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DbetaH), and phenylethanolamine-N-methyltransferase (PNMT). After initial incubation at 37 degrees C with RU486 for 3 days, followed by the addition of gancyclovir for 7 days, Tag-ir was not detectable in most of the surviving chromaffin cells, compared to 100% expression in immortalized loxTsTag/CreR1/RAD cells not treated with RU486 and gancyclovir. The expression of TH, DbetaH, and PNMT was increased after disimmortalization and the ability of disimmortalized cells to synthesize norepinephrine was also significantly increased compared to immortalized cells. When both types of chromaffin cells were transplanted in a model of neuropathic pain and partial nerve injury, both cell grafts were equally able to reverse the behavioral hypersensitivity induced by the injury. The use of Cre/lox site-directed disimmortalization of chromaffin cells that are able to deliver neuroactive molecules offers a novel approach to cell therapy.
Eaton
Mary J
MJ
The Miami Project to Cure Paralysis, University of Miami School of Medicine, 1095 NW 14th Terrace (R-48), Miami, Florida 33136, USA. meaton@miami.edu
Herman
Jean-Paul
JP
Jullien
Nicolas
N
Lopez
Tomas L
TL
Martinez
Miguel
M
Huang
Jian
J
D000818:Animals; D000957:Antigens, Viral, Tumor / Q000235:genetics; D002452:Cell Count; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000166:cytology / Q000187:drug effects / Q000637:transplantation*; D004195:Disease Models, Animal; D004299:Dopamine beta-Hydroxylase / Q000096:biosynthesis; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors / Q000494:pharmacology; D006930:Hyperalgesia / Q000209:etiology / Q000628:therapy; D019426:Integrases / Q000235:genetics; D015735:Mifepristone / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009638:Norepinephrine / Q000096:biosynthesis; D009857:Oncogenes / Q000187:drug effects / Q000235:genetics; D010146:Pain / Q000209:etiology; D059408:Pain Management; D010625:Phenylethanolamine N-Methyltransferase / Q000096:biosynthesis; D051381:Rats; D011920:Rats, Inbred WF; D017207:Rats, Sprague-Dawley; D011995:Recombination, Genetic / Q000187:drug effects; D020426:Sciatic Neuropathy / Q000150:complications / Q000503:physiopathology / Q000628:therapy*; D013696:Temperature; D013937:Thymidine Kinase / Q000235:genetics; D014162:Transfection; D016896:Treatment Outcome; D014446:Tyrosine 3-Monooxygenase / Q000096:biosynthesis; D014764:Viral Proteins / Q000235:genetics
D000957:Antigens, Viral, Tumor; D014764:Viral Proteins; D015735:Mifepristone; D014446:Tyrosine 3-Monooxygenase; D004299:Dopamine beta-Hydroxylase; D010625:Phenylethanolamine N-Methyltransferase; D013937:Thymidine Kinase; C045073:Cre recombinase; D019426:Integrases; D009638:Norepinephrine