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Home > Archive>Volume 29, Issue 8, 2019 >50-54. DOI:10.3969/j. issn. 1671 -7856. 2019. 08. 008
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Establishment of an in vitro culture system for adult marmoset kidney cells and applications of clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 gene editing
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(Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS) &Peking Union Medical Collage (PUMC), Beijing 100021, China)

Clc Number:

R-33

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    Abstract:

    Objective To establish an in vitro culture system of adult marmoset kidney cells, and create a meansfor detecting single guide (sg) RNA cutting activity based on the Cas9 editing system. Methods A growth curve wasinitially determined for adult marmoset kidney cells. The growth curve, transfection reagent and resistance concentrationwere determined. These were then used to co-transfect SIRT1 sgRNA and Cas9 into marmoset cells. Following transfection,the genome was extracted and sequenced. Results The transfection rate of ViaFect was greater than 50% ~ 70%. Theoptimum concentration of puromycin was 4 μg/ mL and that of blasticidin was 8 μg/ mL for cell resistance screening.Sequencing result showed that mutation peaks appeared near the PAM sequence of sgRNA, which proved that sgRNA wassuccessfully introduced mutations into the marmoset genome. Conclusions We successfully established an in vitro culture,transfection and resistance screening system for marmoset kidney cells, which can be used as a reference for gene editing site testing. CRISPR/ Cas9 is suitable for renal cell editing in marmosets.

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History
  • Received:January 23,2019
  • Online: September 12,2019
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