Although retroviral vectors have high efficiency of transgene integration into host cell, there are certain limitations of this approach.

Limitations:

• Low copy number integration.
• Additional steps needed for construction of recombinant retrovirus. 
• Limited size of DNA insert (~<15kb).
• Mosaicism (same individual having two different types of genotypes) of the recovered animals.
• Possible interference of the proviral LTR sequences with the expression of transgene.

6-3.3.3Embryonic stem cell technology:

Embryonic stem (ES) cells are pluripotent stem cells isolated from inner cell mass (ICM) of blastocyst stage of embryo. Use of ES cell mediated transgenic animal production is quite effective specially to generate targeted gene modification at precise location.

The procedure to generate transgenic animals using modified ES cell technology is described in brief below:

• Isolation of pluripotent ES cells from ICM of blastocyst and  culturing of the cells in vitro.
  
  
• Transgene introduction to ES cells  using methods like DNA micro-injection, electroporation, precipitation reaction, transfection etc. DNA transfection to ES cells can be carried out using different vectors like liposome, retroviral vectors etc.
  
  
• Selection of transformed ES cells for either knockin or knockout constructs.
  
  
•  Transformed cells are injected into blastocyst followed by implantation in a surrogate female.
  
  After birth, the offsprings are screened for chimerism. Inbreeding of the genetic chimeras is performed to obtain homozygous transgenic animal carrying both the mutated alleles for that character.