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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

Sexing of sheep known gender using PCR assays based on the amelogenin gene. Male blood samples presented 458 bp and 395 bp bands while female blood samples had only 458 bp bands. Lanes 1–7 are PCR products of genomic DNA from seven female sheep; lanes 9–15 are PCR products of genomic DNA from seven male sheep; lane 8 is 600 bp DNA ladder.
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Fig4: Sexing of sheep known gender using PCR assays based on the amelogenin gene. Male blood samples presented 458 bp and 395 bp bands while female blood samples had only 458 bp bands. Lanes 1–7 are PCR products of genomic DNA from seven female sheep; lanes 9–15 are PCR products of genomic DNA from seven male sheep; lane 8 is 600 bp DNA ladder.

Mentions: After amplification with amelogenin primer, PCR product indicated a 458 bp and 395 bp fragments corresponding to the sheep blood genomic DNA. As expected, male samples presented both bands while female samples had only 458 bp band. For confirming the accuracy of the PCR, 14 blood samples (7 females and 7 males) were detected (Figure 4). All PCR sex determination was in agreement with the actual sexes of the sheep from which blood samples were obtained, indicating that the sexing method based PCR amplified amelogenin gene was 100% reproducible and reliable. 100% (14/14) concordance was obtained using the PCR assay.Figure 4


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)

Sexing of sheep known gender using PCR assays based on the amelogenin gene. Male blood samples presented 458 bp and 395 bp bands while female blood samples had only 458 bp bands. Lanes 1–7 are PCR products of genomic DNA from seven female sheep; lanes 9–15 are PCR products of genomic DNA from seven male sheep; lane 8 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

Fig4: Sexing of sheep known gender using PCR assays based on the amelogenin gene. Male blood samples presented 458 bp and 395 bp bands while female blood samples had only 458 bp bands. Lanes 1–7 are PCR products of genomic DNA from seven female sheep; lanes 9–15 are PCR products of genomic DNA from seven male sheep; lane 8 is 600 bp DNA ladder.
Mentions: After amplification with amelogenin primer, PCR product indicated a 458 bp and 395 bp fragments corresponding to the sheep blood genomic DNA. As expected, male samples presented both bands while female samples had only 458 bp band. For confirming the accuracy of the PCR, 14 blood samples (7 females and 7 males) were detected (Figure 4). All PCR sex determination was in agreement with the actual sexes of the sheep from which blood samples were obtained, indicating that the sexing method based PCR amplified amelogenin gene was 100% reproducible and reliable. 100% (14/14) concordance was obtained using the PCR assay.Figure 4

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