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Advantages of using the CRISPR/Cas9 system of genome editing to investigate male reproductive mechanisms using mouse models.

Young SA, Aitken RJ, Ikawa M - Asian J. Androl. (2015 Jul-Aug)

Bottom Line: Gene disruption technology has long been beneficial for the study of male reproductive biology.Now, it is possible to generate gene-disrupted mouse models in very little time and at very little cost.This Highlight article discusses the application of this technology to study the genetics of male fertility and looks at some of the future uses of this system that could be used to reveal the essential and nonessential genetic components of male reproductive mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Animal Resource Centre for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan, .

ABSTRACT
Gene disruption technology has long been beneficial for the study of male reproductive biology. However, because of the time and cost involved, this technology was not a viable method except in specialist laboratories. The advent of the CRISPR/Cas9 system of gene disruption has ushered in a new era of genetic investigation. Now, it is possible to generate gene-disrupted mouse models in very little time and at very little cost. This Highlight article discusses the application of this technology to study the genetics of male fertility and looks at some of the future uses of this system that could be used to reveal the essential and nonessential genetic components of male reproductive mechanisms.

No MeSH data available.


Related in: MedlinePlus

Conventional versus CRISPR/Cas9 method of gene-disruption. (a) (i and ii): design and construction of a vector; (iii) transfection of ES cells with a vector, followed by multiple rounds of positive and negative selection; (iv) injection of ES cells carrying the mutation into fertilized oocytes; (v) chimeric mice born and bred; and (vi) generation of heterozygous/homozygous mice for analysis. This process takes 1–2 years. (b) (i): design and production of a CRISPR/Cas9 plasmid (the guide sequence is inserted between BbsI restriction enzyme sites); (ii) the CRISPR/Cas9 plasmid is either injected alone if NHEJ is desired, or co-injected with an oligonucleotide if HDR is desired; (iii) plasmid or RNA is injected into the fertilized oocyte; and (iv) the founder generation contains heterozygous/homozygous mice for analysis and proliferation. This process takes only 1–2 months.
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Figure 2: Conventional versus CRISPR/Cas9 method of gene-disruption. (a) (i and ii): design and construction of a vector; (iii) transfection of ES cells with a vector, followed by multiple rounds of positive and negative selection; (iv) injection of ES cells carrying the mutation into fertilized oocytes; (v) chimeric mice born and bred; and (vi) generation of heterozygous/homozygous mice for analysis. This process takes 1–2 years. (b) (i): design and production of a CRISPR/Cas9 plasmid (the guide sequence is inserted between BbsI restriction enzyme sites); (ii) the CRISPR/Cas9 plasmid is either injected alone if NHEJ is desired, or co-injected with an oligonucleotide if HDR is desired; (iii) plasmid or RNA is injected into the fertilized oocyte; and (iv) the founder generation contains heterozygous/homozygous mice for analysis and proliferation. This process takes only 1–2 months.

Mentions: The first step, NHEJ, achieves gene knockout whereas the second HDR step results in very specific mutations being introduced into the host genome (Figure 1).21 The many benefits of CRISPR/Cas9 include cutting out a significant portion of the time to make a knockin/KO animal.21 The usual process of creating a vector, transfecting ES cells, growing and aggregation of ES cells with preimplantation embryos, transplanting to a pseudopregnant female and waiting for pups to be born, then confirming the germline transmission by mating chimeric mice3132 can be lessened as CRISPR/Cas9 is used on fertilized eggs that can be implanted directly into a recipient female mouse.212729 The conventional method can take up to 1–2 years, whereas the CRISPR/Cas9 system takes only 1–2 months to produce homozygous and heterozygous mutant mouse lines (Figure 2).


Advantages of using the CRISPR/Cas9 system of genome editing to investigate male reproductive mechanisms using mouse models.

Young SA, Aitken RJ, Ikawa M - Asian J. Androl. (2015 Jul-Aug)

Conventional versus CRISPR/Cas9 method of gene-disruption. (a) (i and ii): design and construction of a vector; (iii) transfection of ES cells with a vector, followed by multiple rounds of positive and negative selection; (iv) injection of ES cells carrying the mutation into fertilized oocytes; (v) chimeric mice born and bred; and (vi) generation of heterozygous/homozygous mice for analysis. This process takes 1–2 years. (b) (i): design and production of a CRISPR/Cas9 plasmid (the guide sequence is inserted between BbsI restriction enzyme sites); (ii) the CRISPR/Cas9 plasmid is either injected alone if NHEJ is desired, or co-injected with an oligonucleotide if HDR is desired; (iii) plasmid or RNA is injected into the fertilized oocyte; and (iv) the founder generation contains heterozygous/homozygous mice for analysis and proliferation. This process takes only 1–2 months.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492054&req=5

Figure 2: Conventional versus CRISPR/Cas9 method of gene-disruption. (a) (i and ii): design and construction of a vector; (iii) transfection of ES cells with a vector, followed by multiple rounds of positive and negative selection; (iv) injection of ES cells carrying the mutation into fertilized oocytes; (v) chimeric mice born and bred; and (vi) generation of heterozygous/homozygous mice for analysis. This process takes 1–2 years. (b) (i): design and production of a CRISPR/Cas9 plasmid (the guide sequence is inserted between BbsI restriction enzyme sites); (ii) the CRISPR/Cas9 plasmid is either injected alone if NHEJ is desired, or co-injected with an oligonucleotide if HDR is desired; (iii) plasmid or RNA is injected into the fertilized oocyte; and (iv) the founder generation contains heterozygous/homozygous mice for analysis and proliferation. This process takes only 1–2 months.
Mentions: The first step, NHEJ, achieves gene knockout whereas the second HDR step results in very specific mutations being introduced into the host genome (Figure 1).21 The many benefits of CRISPR/Cas9 include cutting out a significant portion of the time to make a knockin/KO animal.21 The usual process of creating a vector, transfecting ES cells, growing and aggregation of ES cells with preimplantation embryos, transplanting to a pseudopregnant female and waiting for pups to be born, then confirming the germline transmission by mating chimeric mice3132 can be lessened as CRISPR/Cas9 is used on fertilized eggs that can be implanted directly into a recipient female mouse.212729 The conventional method can take up to 1–2 years, whereas the CRISPR/Cas9 system takes only 1–2 months to produce homozygous and heterozygous mutant mouse lines (Figure 2).

Bottom Line: Gene disruption technology has long been beneficial for the study of male reproductive biology.Now, it is possible to generate gene-disrupted mouse models in very little time and at very little cost.This Highlight article discusses the application of this technology to study the genetics of male fertility and looks at some of the future uses of this system that could be used to reveal the essential and nonessential genetic components of male reproductive mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Animal Resource Centre for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan, .

ABSTRACT
Gene disruption technology has long been beneficial for the study of male reproductive biology. However, because of the time and cost involved, this technology was not a viable method except in specialist laboratories. The advent of the CRISPR/Cas9 system of gene disruption has ushered in a new era of genetic investigation. Now, it is possible to generate gene-disrupted mouse models in very little time and at very little cost. This Highlight article discusses the application of this technology to study the genetics of male fertility and looks at some of the future uses of this system that could be used to reveal the essential and nonessential genetic components of male reproductive mechanisms.

No MeSH data available.


Related in: MedlinePlus