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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

Primary cultures and identification of PSCs derived from mechanical and enzymatic disaggregation. (A) Enzymes treatment yielded the highest number of cells compared with mechanical disaggregation. (B) Desmin, Pax7 and CD34 were amplified with primers designed to produce an 101, 106 and 858-bp product in the primary satellite cells, respectively. Marker is 600 bp DNA ladder.
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Fig1: Primary cultures and identification of PSCs derived from mechanical and enzymatic disaggregation. (A) Enzymes treatment yielded the highest number of cells compared with mechanical disaggregation. (B) Desmin, Pax7 and CD34 were amplified with primers designed to produce an 101, 106 and 858-bp product in the primary satellite cells, respectively. Marker is 600 bp DNA ladder.

Mentions: To investigate and develop an efficient method to isolate ovine primary satellite cells, collected muscle tissues were digested in three steps by two different enzymes of collagenase for 30 min, trypsin for 30 min followed by digestion with collagenase for 30 min again to induce muscle tissue digestion, and grown in DMEM with 20% FBS and 10% Hours serum. When the same amounts of muscle tissues were used, enzymes treatment was shown to yield the highest number of cells (Figure 1A) compared with mechanical disaggregation.Figure 1


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)

Primary cultures and identification of PSCs derived from mechanical and enzymatic disaggregation. (A) Enzymes treatment yielded the highest number of cells compared with mechanical disaggregation. (B) Desmin, Pax7 and CD34 were amplified with primers designed to produce an 101, 106 and 858-bp product in the primary satellite cells, respectively. Marker is 600 bp DNA ladder.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Primary cultures and identification of PSCs derived from mechanical and enzymatic disaggregation. (A) Enzymes treatment yielded the highest number of cells compared with mechanical disaggregation. (B) Desmin, Pax7 and CD34 were amplified with primers designed to produce an 101, 106 and 858-bp product in the primary satellite cells, respectively. Marker is 600 bp DNA ladder.
Mentions: To investigate and develop an efficient method to isolate ovine primary satellite cells, collected muscle tissues were digested in three steps by two different enzymes of collagenase for 30 min, trypsin for 30 min followed by digestion with collagenase for 30 min again to induce muscle tissue digestion, and grown in DMEM with 20% FBS and 10% Hours serum. When the same amounts of muscle tissues were used, enzymes treatment was shown to yield the highest number of cells (Figure 1A) compared with mechanical disaggregation.Figure 1

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