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Cell culture, sex determination and single cell cloning of ovine transgenic satellite cells in vitro.

Salabi F, Nazari M, Cao WG - J Biol Res (Thessalon) (2014)

Bottom Line: Southern blot results of sex determination were in complete agreement with PCR-amplified bands which confirmed that the HMG box of SRY gene amplified from the ovine genome and that was specific for male.We successfully isolated and cultured sheep primary satellite cells via mechanical and enzymatic disaggregation.The results of sex detection demonstrated that these methods can be applied to detect the sex of primary satellite cells and to determine the sex of sheep embryo prior to produce sheep embryos by somatic cell nuclear transfer technique in vitro.

View Article: PubMed Central - PubMed

Affiliation: Transgenic and Stem Cell Core, Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agricultural Sciences, Beijing, 100193 People's Republic of China.

ABSTRACT

Background: This study was performed to describe the basic methods to isolate and culture of primary satellite cells (PSCs) obtained from 50 to 60-day-old sheep fetuses, single cell cloning of transfected PSCs and sexing of ovine PSCs based on the ZFY/ZFX, amelogenin and high-motility-group (HMG) box sequences.

Results: Three-step enzymatic digestion method increased PSCs isolation from tissue and reduced the damage of cells during long time incubation with enzymes. The results of cloning showed that the 103 and 81 clones (from a total of 184 clones) were derived from feeder and bFGF treatment, respectively. The overall sexing efficiency in the present study was 100%. Southern blot results of sex determination were in complete agreement with PCR-amplified bands which confirmed that the HMG box of SRY gene amplified from the ovine genome and that was specific for male.

Conclusions: We successfully isolated and cultured sheep primary satellite cells via mechanical and enzymatic disaggregation. Our finding demonstrated that use of feeder and addition of bFGF to the culture medium improved cloning efficiency. The results of sex detection demonstrated that these methods can be applied to detect the sex of primary satellite cells and to determine the sex of sheep embryo prior to produce sheep embryos by somatic cell nuclear transfer technique in vitro. Nevertheless, our findings suggested that sex determination of satellite cells base on amelogenin sequence can be accurate, relatively simple, rapid, and inexpensive.

No MeSH data available.


Related in: MedlinePlus

Analysis of GFP expression in primary satellite cells transfected by pEGFP-N1 plasmid. GFP fluorescence in PSCs transfected by pEGFP-N1 plasmid as visualized by fluorescent microscopy. (A) Transfected ovine fetus PSCs 24 hrs, (B) 48 hrs after first transfection (C) and 48 hrs after second transfection. (D) FACS analysis of GFP expression level in a time lapse. The y-axis shows mean of the expression levels and x-axis shows days after transfection. EGFP expression levels for transfected cells and cells number were quantified by measuring the extracellular EGFP by a fluorescence detector for 24 hrs and 48 hrs of first transfection, 3 days post-first transfection and 48 hrs after second transfection on days 1, 2, 5 and 7, respectively. FACS Analysis of GFP expression level for cell number showed that the percentage of transfected cells increased as increasing the transfection time.
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Fig2: Analysis of GFP expression in primary satellite cells transfected by pEGFP-N1 plasmid. GFP fluorescence in PSCs transfected by pEGFP-N1 plasmid as visualized by fluorescent microscopy. (A) Transfected ovine fetus PSCs 24 hrs, (B) 48 hrs after first transfection (C) and 48 hrs after second transfection. (D) FACS analysis of GFP expression level in a time lapse. The y-axis shows mean of the expression levels and x-axis shows days after transfection. EGFP expression levels for transfected cells and cells number were quantified by measuring the extracellular EGFP by a fluorescence detector for 24 hrs and 48 hrs of first transfection, 3 days post-first transfection and 48 hrs after second transfection on days 1, 2, 5 and 7, respectively. FACS Analysis of GFP expression level for cell number showed that the percentage of transfected cells increased as increasing the transfection time.

Mentions: FACS analysis of GFP expression was performed by a cell-based green fluorescent protein (GFP) reporter assay to determine the percentage of GFP-positive cells in each well for characterizing the transfection efficiency and using the GFP reporter system to carry out the cell sorting. At days 1 and 2 of first transfection following by 3 days post double transfection, GFP expression in PSCs was photographed under a fluorescent microscope (Figures 2A-C). Days 1, 2, 5 and 7 of transfection, the transfected pool was tested for GFP expression by flow cytometry and sorted based on the top 0.5% of GFP expression, collected and expanded. The results showed that percentage of transfected cells increased in intensity as the time of transfection with pEGFP-N1 increased. Double transfected cells showed a highest GFP expression level. This result confirmed that the efficiency of transfection was increased by increasing the incubation time and transfection time (Figure 2D).Figure 2


Cell culture, sex determination and single cell cloning of ovine transgenic satellite cells in vitro.

Salabi F, Nazari M, Cao WG - J Biol Res (Thessalon) (2014)

Analysis of GFP expression in primary satellite cells transfected by pEGFP-N1 plasmid. GFP fluorescence in PSCs transfected by pEGFP-N1 plasmid as visualized by fluorescent microscopy. (A) Transfected ovine fetus PSCs 24 hrs, (B) 48 hrs after first transfection (C) and 48 hrs after second transfection. (D) FACS analysis of GFP expression level in a time lapse. The y-axis shows mean of the expression levels and x-axis shows days after transfection. EGFP expression levels for transfected cells and cells number were quantified by measuring the extracellular EGFP by a fluorescence detector for 24 hrs and 48 hrs of first transfection, 3 days post-first transfection and 48 hrs after second transfection on days 1, 2, 5 and 7, respectively. FACS Analysis of GFP expression level for cell number showed that the percentage of transfected cells increased as increasing the transfection time.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4390094&req=5

Fig2: Analysis of GFP expression in primary satellite cells transfected by pEGFP-N1 plasmid. GFP fluorescence in PSCs transfected by pEGFP-N1 plasmid as visualized by fluorescent microscopy. (A) Transfected ovine fetus PSCs 24 hrs, (B) 48 hrs after first transfection (C) and 48 hrs after second transfection. (D) FACS analysis of GFP expression level in a time lapse. The y-axis shows mean of the expression levels and x-axis shows days after transfection. EGFP expression levels for transfected cells and cells number were quantified by measuring the extracellular EGFP by a fluorescence detector for 24 hrs and 48 hrs of first transfection, 3 days post-first transfection and 48 hrs after second transfection on days 1, 2, 5 and 7, respectively. FACS Analysis of GFP expression level for cell number showed that the percentage of transfected cells increased as increasing the transfection time.
Mentions: FACS analysis of GFP expression was performed by a cell-based green fluorescent protein (GFP) reporter assay to determine the percentage of GFP-positive cells in each well for characterizing the transfection efficiency and using the GFP reporter system to carry out the cell sorting. At days 1 and 2 of first transfection following by 3 days post double transfection, GFP expression in PSCs was photographed under a fluorescent microscope (Figures 2A-C). Days 1, 2, 5 and 7 of transfection, the transfected pool was tested for GFP expression by flow cytometry and sorted based on the top 0.5% of GFP expression, collected and expanded. The results showed that percentage of transfected cells increased in intensity as the time of transfection with pEGFP-N1 increased. Double transfected cells showed a highest GFP expression level. This result confirmed that the efficiency of transfection was increased by increasing the incubation time and transfection time (Figure 2D).Figure 2

Bottom Line: Southern blot results of sex determination were in complete agreement with PCR-amplified bands which confirmed that the HMG box of SRY gene amplified from the ovine genome and that was specific for male.We successfully isolated and cultured sheep primary satellite cells via mechanical and enzymatic disaggregation.The results of sex detection demonstrated that these methods can be applied to detect the sex of primary satellite cells and to determine the sex of sheep embryo prior to produce sheep embryos by somatic cell nuclear transfer technique in vitro.

View Article: PubMed Central - PubMed

Affiliation: Transgenic and Stem Cell Core, Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agricultural Sciences, Beijing, 100193 People's Republic of China.

ABSTRACT

Background: This study was performed to describe the basic methods to isolate and culture of primary satellite cells (PSCs) obtained from 50 to 60-day-old sheep fetuses, single cell cloning of transfected PSCs and sexing of ovine PSCs based on the ZFY/ZFX, amelogenin and high-motility-group (HMG) box sequences.

Results: Three-step enzymatic digestion method increased PSCs isolation from tissue and reduced the damage of cells during long time incubation with enzymes. The results of cloning showed that the 103 and 81 clones (from a total of 184 clones) were derived from feeder and bFGF treatment, respectively. The overall sexing efficiency in the present study was 100%. Southern blot results of sex determination were in complete agreement with PCR-amplified bands which confirmed that the HMG box of SRY gene amplified from the ovine genome and that was specific for male.

Conclusions: We successfully isolated and cultured sheep primary satellite cells via mechanical and enzymatic disaggregation. Our finding demonstrated that use of feeder and addition of bFGF to the culture medium improved cloning efficiency. The results of sex detection demonstrated that these methods can be applied to detect the sex of primary satellite cells and to determine the sex of sheep embryo prior to produce sheep embryos by somatic cell nuclear transfer technique in vitro. Nevertheless, our findings suggested that sex determination of satellite cells base on amelogenin sequence can be accurate, relatively simple, rapid, and inexpensive.

No MeSH data available.


Related in: MedlinePlus