Effect of the Compaction and the Size of DNA on the Nuclear Transfer Efficiency after Microinjection in Synchronized Cells

نویسندگان

  • Hidetaka Akita
  • Dai Kurihara
  • Marco Schmeer
  • Martin Schleef
  • Hideyoshi Harashima
  • Keiji Itaka
چکیده

The nuclear transfer process is one of the critical rate-limiting processes in transgene expression. In the present study, we report on the effect of compaction and the size of the DNA molecule on nuclear transfer efficiency by microinjection. A DNA/protamine complex- or variously-sized naked DNA molecules were injected into the cytoplasm or nucleus of synchronized HeLa cells. To evaluate the nuclear transfer process, a nuclear transfer score (NT score), calculated based on transgene expression after cytoplasmic microinjection divided by that after nuclear microinjection, was employed. The compaction of DNA with protamine decreased the NT score in comparison with the injection of naked DNA when the N/P ratio was increased to >2.0. Moreover, when naked DNA was microinjected, gene expression increased in parallel with the size of the DNA in the following order: minicircle DNA (MC07.CMV-EGFP; 2257 bp) > middle-sized plasmid DNA (pBS-EGFP; 3992 bp) > conventional plasmid DNA (pcDNA3.1-EGFP; 6172 bp), while the level of gene expression was quite comparable among them when the DNAs were injected into the nucleus. The above findings suggest that the intrinsic size of the DNA molecule is a major determinant for nuclear entry and that minicircle DNA has a great advantage in nuclear transfer.

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

The Effect of Linear PEI on Characteristics and Transfection Efficiency of PEI-Based Cationic Nanoliposomes

The development of efficient and safe carrier system to transfer DNA into cells is essential in non-viral gene therapy. The aim of the present study was to evaluate the effect of linear polyetheneimine (lPEI) (2500 Da) on the physicochemical and biological properties of lipopolyplexes constructed from liposomes and lPEI. Materials and Methods Different lipopolymers were synthesized from lPEI ...

متن کامل

Efficiency of Ovine Fibroblast or Cumulus Cells For Somatic Cell Nuclear Transfer in Sheep

Purpose: Despite remarkable progresses have been achieved in the field of somatic cell nuclear transfer (SCNT), there is little information regarding the effect of donor cell type on the efficiency mammalian somatic cell cloning in vitro. This study compared in vitro developmental competency of sheep enucleated oocytes reconstructed with either fibroblast or cumulus cells. Material and methods...

متن کامل

Effect of different mass ratio of PLA: PEG segments in PLA-PEG-PLA copolymers on the physicochemical characterization and DNA release profile

Background: Adapting controlled release technologies to the delivery of DNA has the great potential to overcome extracellular barriers that limit gene delivery. This study investigates the effect of different mass ratio of PLA: PEG in the various tri block poly (lactic acid)-poly (ethylene glycol) - Poly (lactic acid) copolymer (PLA-PEG-PLA) on the properties of the resulting nanoparticles. Me...

متن کامل

I-5: Fifteen Years after Dolly: The Perspectives on Cellular Reprogramming

s:1202:"It is a truly amazing time to developmental biology. During recent decades, three important breakthroughs have been developed: (i) isolation of stem cells from embryo, (ii) animal cloning by nuclear transfer (NT), and (iii) and induced pluripotent stem cells (iPS). Considering these three approaches of "Cellular Reprogramming", it seems that the required elements for cell therapy now ex...

متن کامل

I-12: Nuclear Reprogramming in Bovin Somatic Cell Nuclear Transfer

Somatic cell nuclear transfer (SCNT or cloning) returns a differentiated cell to a totipotent status; a process termed nuclear reprogramming. Reproductive cloning has potential applications in both agriculture and biomedicine, but is limited by low efficiency. To understand the deficiencies of nuclear reprogramming, our research has focused on both candidate genes and global gene expression pat...

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

عنوان ژورنال:

دوره 7  شماره 

صفحات  -

تاریخ انتشار 2015