Germline modification of domestic animals

Genetically-modified domestic animal models are of increasing significance in biomedical research and agriculture. As authentic ES cells derived from domestic animals are not yet available, the prevailing approaches for engineering genetic modifications in those animals are pronuclear microinjection and somatic cell nuclear transfer (SCNT, also known as cloning). Both pronuclear microinjection and SCNT are inefficient, costly, and time-consuming. In animals produced by pronuclear microinjection, the exogenous transgene is usually inserted randomly into the genome, which results in highly variable expression patterns and levels in different founders. Therefore, significant efforts are required to generate and screen multiple founders to obtain animals with optimal transgene expression. For SCNT, specific genetic modifications (both gain-of-function and loss-of-function) can be engineered and carefully selected in the somatic cell nucleus before nuclear transfer. SCNT has been used to generate a variety of genetically modified animals such as goats, pigs, sheep and cattle; however, animals resulting from SCNT frequently suffer from developmental abnormalities associated with incomplete nuclear reprogramming. Other strategies to generate genetically-modified animals rely on the use of the spermatozoon as a natural vector to introduce genetic material into the female gamete. This sperm mediated DNA transfer (SMGT) combined with intracytoplasmatic sperm injection (ICSI) has relatively high efficiency and allows the insertion of large DNA fragments, which, in turn, enhance proper gene expression. An approach currently being developed to complement SCNT for producing genetically modified animals is germ cell transplantation using genetically modified male germline stem cells (GSCs). This approach relies on the ability of GSCs that are genetically modified in vitro to colonize the recipient testis and produce donor derived sperm upon transplantation.

Germline modification of domestic animals

Genetically-modified domestic animal models are of increasing significance in biomedical research and agriculture. As authentic ES cells derived from domestic animals are not yet available, the prevailing approaches for engineering genetic modifications in those animals are pronuclear microinjection and somatic cell nuclear transfer (SCNT, also known as cloning). Both pronuclear microinjection and SCNT are inefficient, costly, and time-consuming. In animals produced by pronuclear microinjection, the exogenous transgene is usually inserted randomly into the genome, which results in highly variable expression patterns and levels in different founders. Therefore, significant efforts are required to generate and screen multiple founders to obtain animals with optimal transgene expression. For SCNT, specific genetic modifications (both gain-of-function and loss-of-function) can be engineered and carefully selected in the somatic cell nucleus before nuclear transfer. SCNT has been used to generate a variety of genetically modified animals such as goats, pigs, sheep and cattle; however, animals resulting from SCNT frequently suffer from developmental abnormalities associated with incomplete nuclear reprogramming. Other strategies to generate genetically-modified animals rely on the use of the spermatozoon as a natural vector to introduce genetic material into the female gamete. This sperm mediated DNA transfer (SMGT) combined with intracytoplasmatic sperm injection (ICSI) has relatively high efficiency and allows the insertion of large DNA fragments, which, in turn, enhance proper gene expression. An approach currently being developed to complement SCNT for producing genetically modified animals is germ cell transplantation using genetically modified male germline stem cells (GSCs). This approach relies on the ability of GSCs that are genetically modified in vitro to colonize the recipient testis and produce donor derived sperm upon transplantation.(AU)

Effects of culture medium and substrate on attachment of in vitro produced bovine embryos

The study of large animal embryonic stem cells (ESCs) in vitro has implications for the understanding of lineage differentiation and transgenesis. The first step for ESC derivation is the attachment of the embryo to a substrate on which they can form outgrowths. However, the culture conditions for large animal embryo attachment and ESC derivation have not been studied extensively. Defining culture conditions for embryo attachment such as culture medium and substrate is an important first step for derivation of inner cell mass-derived stem cells. The aim of this study was to compare different types of culture media and substrates for their ability to support attachment of in vitro produced bovine embryos in culture. Bovine embryos were produced in vivo following established protocols. Blastocysts formed on day 8 after fertilization were transferred to 12-well culture plates containing different types of culture media (Dulbecco's Modified Eagle Medium, DMEM or Medium 199, M199) and substrates [bovine fetal fibroblasts, goat fetal fibroblasts, mouse embryonic fibroblasts (STO) or non-cellular substrates (gelatin, laminin, fibronectin)]. Percentage of attached embryos and number of days since fertilization required for attachment were recorded. Bovine blastocysts preferrably attached to feeder cells rather than non-cellular substrates and there was an interact ion of feeder cell type and culture medium used. Therefore, the choice of both feeder cell type and culture medium has to be considered when optimizing conditions to derive cell lines from bovine embryos.

Effects of culture medium and substrate on attachment of in vitro produced bovine embryos

The study of large animal embryonic stem cells (ESCs) in vitro has implications for the understanding of lineage differentiation and transgenesis. The first step for ESC derivation is the attachment of the embryo to a substrate on which they can form outgrowths. However, the culture conditions for large animal embryo attachment and ESC derivation have not been studied extensively. Defining culture conditions for embryo attachment such as culture medium and substrate is an important first step for derivation of inner cell mass-derived stem cells. The aim of this study was to compare different types of culture media and substrates for their ability to support attachment of in vitro produced bovine embryos in culture. Bovine embryos were produced in vivo following established protocols. Blastocysts formed on day 8 after fertilization were transferred to 12-well culture plates containing different types of culture media (Dulbecco's Modified Eagle Medium, DMEM or Medium 199, M199) and substrates [bovine fetal fibroblasts, goat fetal fibroblasts, mouse embryonic fibroblasts (STO) or non-cellular substrates (gelatin, laminin, fibronectin)]. Percentage of attached embryos and number of days since fertilization required for attachment were recorded. Bovine blastocysts preferrably attached to feeder cells rather than non-cellular substrates and there was an interact ion of feeder cell type and culture medium used. Therefore, the choice of both feeder cell type and culture medium has to be considered when optimizing conditions to derive cell lines from bovine embryos.(AU)