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SLIM microscopy allows for visualization of DNA-containing liposomes designed for sperm-mediated gene transfer in cattle

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Abstract

Naked DNA has been shown to bind naturally to the sperm, a method called sperm-mediated gene transfer (SMGT). Based on these observations, we examined the efficiency of exogenous DNA binding to sperm using liposomes. In this experiment, we analyzed methods to select frozen-thawed bovine sperm, and evaluated the binding of exogenous DNA to those sperm. To determine the optimal selection method, we used Computer-Assisted Sperm Analysis (CASA). Percoll or Swim-Up were used to select sperm, followed by incubation up to 3 h with the liposome-DNA complexes. The samples were collected after 1 h and after 3 h. We used enhanced green fluorescent protein (eGFP) in combination with the liposomes as a marker for exogenous DNA binding. Five treatments per selection method were analyzed: (1) no incubation, no liposomes and no DNA, (2) incubation with no liposomes and no DNA, (3) incubation with liposomes and no DNA, (4) incubation with liposomes and 1 µg of DNA and (5) incubation with liposomes and 10 µg of DNA. The CASA results for total motility and rapid motility were statistically significant (P < 0.01) between the control and the other treatments in the Percoll group as opposed to Swim-Up. Swim-Up was therefore chosen as the optimal selection method. In order to determine if the liposome-DNA complex had bound to sperm, real time PCR was used to detect GFP DNA and images of the sperm were analyzed using the Spatial Light Interference Microscopy (SLIM). SLIM confirmed the presence of liposomes on the sperm head and tail.

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Funding

Funding was provided by National Institute of Food and Agriculture (Grant No. Multi-State W3171-ILLU-538-347).

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Author notes

  1. Marcello Rubessa and Samantha N. Lotti are contributed equally to this work.

Authors and Affiliations

  1. Department of Animal Sciences, University of Illinois, Urbana-Champaign, IL, 61801, USA

    Marcello Rubessa, Samantha N. Lotti & Mathew B. Wheeler

  2. Carl R. Woese Institute for Genomic Biology, Univ. of Illinois at Urbana-Champaign, Urbana-Champaign, USA

    Mathew B. Wheeler

  3. Department of Bioengineering, University of Illinois, Urbana, IL, 61801, USA

    Gabriel Popescu & Mathew B. Wheeler

  4. Quantitative Light Imaging Laboratory, Department of Electrical and Computer Engineering, Beckman Institute of Advanced Science and Technology, Urbana, USA

    Mikhail E. Kandel & Gabriel Popescu

Authors
  1. Marcello Rubessa
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  2. Samantha N. Lotti
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  3. Mikhail E. Kandel
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  4. Gabriel Popescu
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  5. Mathew B. Wheeler
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Corresponding author

Correspondence to Mathew B. Wheeler.

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Conflict of interest

The authors (M.R., S.N.L., M.E.K. and M.B.W.) declare that they have no conflict of interest. G.P. has financial interest in Phi Optics, Inc., a company developing quantitative phase imaging technology for materials and life science applications.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors. We did not used any live animals for these studies. The frozen semen used was from a commercial semen vendor. We purchased the semen used it these studies and had no contact with the bulls used for the in vitro fertilization. Our University does not require IACUC (animal care and use) protocols when using purchased tissues or abattoir materials. We therefore see no ethics issues with the present study.

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Rubessa, M., Lotti, S.N., Kandel, M.E. et al. SLIM microscopy allows for visualization of DNA-containing liposomes designed for sperm-mediated gene transfer in cattle. Mol Biol Rep 46, 695–703 (2019). https://doi.org/10.1007/s11033-018-4525-9

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  • DOI: https://doi.org/10.1007/s11033-018-4525-9

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