Germline transmission

ES cellsb Simple Inexpensive None None Need to make germline transmitting chimeras Upon germline transmission only half the ES derived offspring will be heterozygotes ES cells carry tissue culture associated genetic damage Not scalable [Pg.28]

Lois, C., Hong, E.J., Pease, S., Brown, E.J., and Baltimore, D. (2002) Germline transmission and tissue-specific expression of trans-... 

Hadjantonakis, A.K., Gertsenstein, M., Ikawa, M., Okabe, M., and Nagy, A. (1998) Generating green fluorescent mice by germline transmission of green fluorescent ES cells. Mech. Dev. 76, 79-90. 

K. A. High. 2001. Lack of germline transmission of vector sequences following systemic administration of recombinant AAV-2 vector in males. Mol. Ther. 4 586-592. 

Gene therapy, DNA vaccines, genetically modified tissue or cell-based products. EMEA/273974/05 Draft Annex on nonclinical testing for inadvertent germline transmission of gene transfer vectors [36] 2005... 

Lois C, Hong EJ, Pease S, et al. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science. 2002 295(5556) 868-872. 

Tomizuka, K., Yoshida, H., Uejima, H., Ku-goh, H., Sato, K., Ohguma, A., Hayasaka, M., Hanaoka, K., Oshimura, M., Ishida, 1. Functional expression and germline transmission of a human chromosome fragment in chimae-ric mice. Nat. Genet. 1997, 36, 133-143. 

Fig. 1. Germline transmission from one generation to the next generation in sexual reproduction system. In most vertebrate species, the primary cxx ytes (or immature oocytes) in ovarian tissue pauses their cell cycle at prophase of the first meiosis, resumes the meiosis in response to hormonal signals, re-pauses at metaphase of the second meiotic cell cycle as the secondary oocytes (or mature oocytes), and are subject to ovulation and fertilization. Upon fertilization, eggs undergo a series of extracellular and intracellular reactions/changes, collectively called egg activation that triggers the initiation of development or early embryogenesis. A similar mechanism, although not identical in several sp>ecies, has been shown to be involved in sexual reproduction system in a diverse array of animal species and maybe in some algae and plants.

Briefly, germline transmission of the primary loxP-flanked DNA sequence is achieved. 

Carmell MA, Zhang L, Conklin DS, Hannon GJ, Rosenquist TA (2003) Germline transmission of RNAi in mice. Nat Struct Biol 10 91-92. 

The first transgene to be delivered into medaka embryos was the 6-crystalline gene of chicken (10) resulting in transient mosaic expression of the transgene. In 1988, Stuart McMurray, and Westerfield reported the first successful germline transmission and subsequent expression of a transgene on microinjection in a teleost genetic model system (zebrafish (12)). 

Raz and colleagues used Tc3 from C. elegans to stably introduce a reporter construct containing GFP into zebrafish embryos by coinjechon of the Tc3 transposase mRNA together with the reporter flanked by inverted repeats (46). In one line, they could show transposon-mediated integrahon, expression of the reporter construct and germline transmission. 

Collas P, Alestrom P (1998) Nuclear localization signals enhance germline transmission of a transgene in zebrafish. Transgenic Res 7 303-309. 

Once thawed, blastomeres cells can be transplanted into a recipient embryo, leading to the formation of a chimera. The purpose of such transplantation is so injected cells contribute to formation of the germline. Chimera production with cryopreserved cells has been successfully reported in rainbow trout (Takeuchi et al, 2001,2004), together with successful germline transmission, although transmission rates were very low. Germline transmission rates were improved after the recipient cells were weakened by irradiation (Joly et al, 1999). In zebrafish, only two adult chimaeric specimens were obtained from a total of 47 injected embryos... 

Cardona-Costa et /., 2009), and no germline transmission was achieved. It has also been reported that cryopreservation can disrupt normal gene expression patterns in zebrafish embryonic blastomeres this disruption may have had detrimental effects on embryo development (Lin et al, 2009a). 

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