Whole Animal In Vivo Imaging After Transient, Nonviral Gene delivery to the Rat Central Nervous System

Abstract

We previously showed that a vector:lipid delivery system, comprised of a plasmid DNA vector and cationic lipid (lipoplex), when injected into the cerebrospinal fluid (CSF) of rats can deliver reporter genes in vivo efficiently and with widespread expression to the Central Nervous System (CNS). To further characterize this delivery system, we now present experiments that demonstrate the in vivo time-to-peak expression of the reporter gene, firefly luciferase. We infused a formulated lipoplex containing the lipid MLRI [dissymmetric myristoyl (14:0) and lauroyl (12:1) rosenthal inhibitor–substituted compound formed from the tetraalkylammonium glycerol–based DORI] and pNDluc, a luciferase vector, into CSF in the cisterna magna (CM) of the rat. Luciferase activity was followed over time by bioluminescence imaging after injection of luciferin. Our results show that luciferase activity in the CNS of rats is widespread, peaks 72 hours after injection into CM and can be detected in vivo for at least 7–10 days after peak expression. We further show that in contrast to injection into CSF, enzyme activity is not widely distributed after injection of the vector into brain parenchyma, emphasizing the importance of CSF delivery to achieve widespread vector distribution. Finally, we confirm the distribution of firefly luciferase in brain by immunohistochemical staining from an animal that was euthanized at the peak of enzyme expression.

Retracted: Ascorbic Acid Improves Embryonic Cardiomyoblast Cell Survival and Promotes Vascularization in Potential Myocardial Grafts In Vivo

“…They were placed in the chamber in supine position and peak luciferase activity was detected by imaging the animals for 40 min with 2-min acquisition separated by 2-min intervals. 17 The same rats were scanned repeatedly postimplantation at days 1, 3, and 6. Regions of interest indicating the locations of most intense signals were created using Living Imaging Software version 2.6.…”

“…[15][16][17] Briefly, rats were anesthetized with 2% isoflurane, and d-luciferin was administered intraperitoneally at a dose of 150 mg=kg of body weight. They were placed in the chamber in supine position and peak luciferase activity was detected by imaging the animals for 40 min with 2-min acquisition separated by 2-min intervals.…”

Non-Viral Nucleic Acid Delivery Strategies to the Central Nervous System

“…After injection into the cisterna magma of rats, liposomes delivered luciferase plasmid throughout the brain that was still detectable 7–10 days later (Hauck et al, 2008). Interestingly, when the same system was directly injected into the parenchyma, luciferase expression was not as distributed.…”

Enhancement of reporter gene detection sensitivity by insertion of specific mini-peptide-coding sequences

“…The use of gene therapy ranges from the utilization of reporter genes for noninvasive monitoring and gene distribution1–3 to the application of therapeutic genes to treat several diseases such as cancer4–7 and blood disorders8, 9. Advancements in the development of both viral and non-viral delivery systems for gene therapy result in continuous improvements in the efficacy of gene therapy, but several barriers remain which are blocking gene therapy from becoming widely successful.…”

“…SEAP is used for a variety of in vitro and in vivo assays including interferon activity10, efficacy of antidepressants11, and endoplasmic reticulum stress12, 13. With current strategies, the low level of gene expression of SEAP as well as other reporter genes affects the use of reporter genes and limits their uses to in vitro and small animal models1–3, 12, 14. Similarly, therapeutic applications of gene therapy are hindered by having trouble acquiring therapeutically significant levels of the gene products15, 16.…”